Chimeric antigen receptor t cell therapy

ABSTRACT

Provided herein are methods for preparing, producing, processing, culturing, isolating, or making cells suitable for immune or cell therapy, and for their use in cell therapy.

FIELD

The present application relates to CAR-T cells, methods of making them,and methods of using them to treat cancer.

BACKGROUND

Human cancers are by their nature comprised of normal cells that haveundergone a genetic or epigenetic conversion to become abnormal cancercells. Cancer cells express proteins and other antigens that aredistinct from those expressed by normal cells. These aberrant tumorantigens may be used by the body's innate immune system to specificallytarget and kill cancer cells. However, cancer cells employ variousmechanisms to prevent immune cells, such as T and B lymphocytes, fromsuccessfully targeting cancer cells. Human T cell therapies rely onex-vivo-enriched or modified human T cells to target and kill cancercells in a subject, e.g., a patient. Various technologies have beendeveloped to prepare T cell populations with enriched concentrations ofnaturally occurring T cells capable of targeting a tumor antigen, removecirculating tumor cells, and/or genetically modifying T cells tospecifically target a known cancer antigen, thus producing populationsof chimeric antigen receptor (CAR)-T cells for cancer therapy. Some ofthese therapies have shown promising effects on tumor size and patientsurvival.

SUMMARY

Any aspect or embodiment described herein may be combined with any otheraspect or embodiment as disclosed herein. While the present inventionhas been described in conjunction with the detailed description thereof,the description is intended to illustrate and not limit the scope of thepresent invention, which is partially defined by the scope of theappended claims. Other aspects, advantages, and modifications are withinthe scope of the following embodiments/claims.

Embodiment 1. A method for treating mantle cell lymphoma (MCL) or B cellALL in a subject in need thereof comprising administering to the subjecta therapeutically effective amount of a T cell product comprisingautologous T cells expressing an anti-CD19 chimeric antigen receptor(CAR).

Embodiment 2. The method of embodiment 1, wherein the MCL and B cell ALLare relapsed or refractory MCL and B cell ALL, optionally wherein theMCL is classical, blastoid, and pleomorphic MCL.

Embodiment 3. The method of any one of embodiments 1 and 2, wherein theMCL and B cell ALL is refractory to, or has relapsed following, one ormore of chemotherapy, radiotherapy, immunotherapy (including a T celltherapy and/or treatment with an antibody or antibody-drug conjugate),an autologous stem cell transplant, or any combination thereof.

Embodiment 4. The method of any one of embodiments 1 through 3, whereinthe subject has received 1-5 prior treatments, optionally wherein atleast one of the prior treatments is selected from autologous SCT,anti-CD20 antibody, anthracycline- or bendamustine-containingchemotherapy, and/or a Bruton Tyrosine Kinase inhibitor (BTKi).

Embodiment 5. The method of embodiment 4, wherein the BTKi is ibrutinibor acalabrutinib.

Embodiment 6. The method of any one of embodiments 1 through 5, whereinR/R B cell ALL is defined as refractory to first-line therapy (i.e.,primary refractory), relapsed ≤12 months after first remission, relapsedor refractory after ≥2 prior lines of systemic therapy, or relapsedafter allogeneic stem cell transplant (SCT), optionally, wherein thesubject is required to have ≥5% bone marrow blasts, an EasternCooperative Oncology Group performance status of 0 or 1, and/or adequaterenal, hepatic, and cardiac function.

Embodiment 7. The method of any one of embodiments 1 through 6, whereinif the B cell ALL subject has received prior blinatumomab, the subjectis required to have leukemic blasts with CD19 expression ≥90%.

Embodiment 8. The method of any one of embodiments 1 through 7, whereinthe subject receives bridging therapy after leukapheresis and beforeconditioning/lymphodepleting chemotherapy.

Embodiment 9. The method of any one of embodiments 1 through 8, whereinthe MCL subject receives a lymphodepleting chemotherapy regimen ofcyclophosphamide 500 mg/m2 intravenously and fludarabine 30 mg/m2intravenously, both given on each of the fifth, fourth, and third daysbefore T cell infusion.

Embodiment 10. The method of any one of embodiments 1 through 9, whereinthe B cell ALL subject receives a lymphodepleting regimen of fludarabineintravenous (IV) 25 mg/m2/day on each of the fourth, third, second daysbefore T cell infusion, and cyclophosphamide IV 900 mg/m2/day on thesecond day before infusion.

Embodiment 11. The method of any one of embodiments 8 through 10,wherein the MCL bridging therapy is selected from dexamethasone (e.g.,20-40 mg or equivalent PO or IV daily for 1-4 days); methylprednisolone,ibrutinib (e.g., 560 mg PO daily), and/or acalabrutinib (e.g, 100 mg POtwice daily); an immunomodulator; R-CHOP, bendamustine; alkylatingagents; and/or platinum-based agents, wherein the bridging therapy isadministered after leukapheresis and completed in, for example, 5 daysor less before conditioning chemotherapy.

Embodiment 12. The method of any one of embodiments 8 through 10,wherein the B cell ALL subject may receive any one or more of thefollowing bridging chemotherapy regimens:

Predefined Bridging Chemotherapy Regimens Attenuated VAD Vincristinenon-liposomal (1-2 mg IV weekly) or liposomal (2.25 mg/m² IV weekly),and dexamethasone 20-40 mg IV or PO daily × 3-4 days per week. Optionaldoxorubicin 50 mg/m² IV × 1 (first week only) Mercaptopurine 50-75mg/m²/day by mouth (administer at bedtime on an empty (6-MP) stomach toimprove absorption) Hydroxyurea Doses titrated between 15-50 mg/kg/day(rounded to the nearest 500 mg capsule and given as a single daily oraldose on a continuous basis) DOMP Dexamethasone 6 mg/m²/day PO (or IV)divided BID days 1-5, vincristine 1.5 mg/m² (maximum dose 2 mg) IV onday 1, methotrexate 20 mg/m² PO weekly, 6-MP 50-75 mg/m²/day PO dailyAttenuated Fludarabine 30 mg/m² IV days 1-2, cytarabine 2 g/m² IV days1-2, FLAG/FLAG-IDA G-CSF 5 μg/kg SC or IV starts on day 3 and cancontinue until day before the start of conditioning chemotherapy. Withor without idarubicin 6 mg/m² IV days 1-2 Mini-hyper CVAD Course A:Cyclophosphamide 150 mg/m² every 12 h × 3 days, (courses A and/ordexamethasone 20 mg/d IV or PO daily days 1-4 and 11-14, B) vincristine2 mg IV × 1 Course B: methotrexate 250 mg/m² IV over 24 hours on day 1,cytarabine 0.5 g/m² IV every 12 hours × 4 doses on days 2 and 3

Embodiment 13. The method of any one of embodiments 1 through 12,wherein the T cell product comprises CD4+ and CD8+ CAR T cells that areprepared from peripheral blood mononuclear cells (PBMCs) by positiveenrichment and consequent partial or complete depletion of circulatingcancer cells.

Embodiment 14. The method of embodiment 13, wherein the PBMC areenriched for T cells by positive selection for CD4+ and CD8+ cells,activated with anti-CD3 and anti-CD28 antibodies in the presence ofIL-2, and then transduced with a replication-incompetent viral vectorcontaining FMC63-28Z CAR, a chimeric antigen receptor (CAR) comprisingan anti-CD19 single-chain variable fragment (scFv), CD28 and CD3-zetadomains.

Embodiment 15. The method of any one of embodiments 13 and 14, whereinthe T cell product comprises fewer cancer cells than a T cell productcomprising T cells from a leukapheresis product that have not beenpositively selected for CD4+ and CD8+ T cells.

Embodiment 16. The method of any one of embodiments 13 through 15,wherein the T cell product has other superior product attributesrelative to a T cell product comprising T cells from a leukapheresisproduct that have not been positively selected/enriched for CD4+ andCD8+ T cells.

Embodiment 17. The method of embodiment 16, wherein the superior productattributes are selected from increased percentage of CDRA45+CCR7+(naïve-like) T cells, decreased percentage of differentiated Tcells, increased percentage of CD3+ cells, decreased IFN-gammaproduction, decreased percentage of CD3− cells.

Embodiment 18. The method of any one of embodiments 1 through 17,wherein the MCL subject is administered one or more doses of 1.8×106,1.9×106, or 2×106 CAR positive viable T cells per kg body weight, with amaximum of 2×108 CAR positive viable T cells (for patients 100 kg andabove) and the B cell ALL subject is administered 0.5×106, 1×106, or2×106 CAR positive viable T cells per kg body weight, with a maximum of2×108 CAR positive viable T cells (for patients 100 kg and above).

Embodiment 19. The method of any one of embodiments 1 through 17,wherein if the subject has achieved complete response to the firstinfusion, the subject may receive a second infusion of anti-CD19 CAR Tcells, if progressing following >3 months of remission, provided CD19expression has been retained and neutralizing antibodies against the CARare not suspected, wherein response is assessed using the Luganoclassification.

Embodiment 20. The method of any one of embodiments 1 through 19,wherein the subject is monitored for signs and symptoms of cytokinerelease syndrome (CRS) and neurologic toxicity after T celladministration.

Embodiment 21. The method of embodiment 20, wherein the subject ismonitored daily for at least seven days, preferably for four weeks,following infusion for signs and symptoms of CRS and neurologictoxicity.

Embodiment 22. The method of any one of embodiments 20 and 21, whereinthe signs or symptoms associated with CRS include fever, chills,fatigue, tachycardia, nausea, hypoxia, and hypotension and the signs orsymptoms associated with neurologic events include encephalopathy,seizures, changes in level of consciousness, speech disorders, tremors,and confusion.

Embodiment 23. The method of any one of embodiments 20 through 22,wherein cytokine release syndrome in MCL subjects is managed inaccordance with the following protocol:

CRS Grade Tocilizumab Corticosteroids Grade 1 If not improving after 24Not applicable. Symptoms require hours, administer tocilizumabsymptomatic treatment only 8 mg/kg intravenously over 1 (e.g., fever,nausea, fatigue, hour (not to exceed 800 mg). headache, myalgia,malaise). Grade 2 Administer tocilizumab 8 Manage per Grade 3 Symptomsrequire and mg/kg intravenously over 1 if no improvement respond tomoderate hour (not to exceed 800 mg). within 24 hours afterintervention. Repeat tocilizumab every 8 starting tocilizumab. Oxygenrequirement less than hours as needed if not If improving, taper 40%FiO2 or hypotension responsive to intravenous fluids corticosteroids.responsive to fluids or low or increasing supplemental dose of onevasopressor or oxygen. Limit to a maximum of Grade 2 organ toxicity. 3doses in a 24-hour period; maximum total of 4 doses if no clinicalimprovement in the signs and symptoms of CRS. If improving, discontinuetocilizumab. Grade 3 Per Grade 2 Administer Symptoms require andmethylprednisolone respond to aggressive 1 mg/kg intravenouslyintervention. twice daily or Oxygen requirement greater equivalent thanor equal to 40% FiO2 or dexamethasone (e.g., hypotension requiring high-10 mg intravenously dose or multiple vasopressors every 6 hours) untilor Grade 3 organ toxicity or Grade 1, then taper Grade 4 transaminitis.corticosteroids. If improving, manage as Grade 2. If not improving,manage as Grade 4. Grade 4 Per Grade 2 Administer Life-threateningsymptoms. methylprednisolone Requirements for ventilator 1000 mg supportor continuous veno- intravenously per venous hemodialysis day for 3days. (CVVHD), or Grade 4 organ If improving, taper toxicity (excludingcorticosteroids, and transaminitis). manage as Grade 3. If notimproving, consider alternate immunosuppressants.

Embodiment 24. The method of any one of embodiments 20 through 23,wherein neurologic toxicity in MCL subjects is managed in accordancewith the following protocol:

Grading Assessment Concurrent CRS No Concurrent CRS Grade 2 Administertocilizumab per Administer dexamethasone embodiment 15 for management 10mg intravenously every of Grade 2 CRS. 6 hours until the event is GradeIf not improving within 24 hours after 1 or less, then taper startingtocilizumab, administer corticosteroids. dexamethasone 10 mgintravenously every 6 hours until the event is Grade 1 or less, thentaper corticosteroids. If still not improving, manage as Grade 3.Consider non-sedating anti-seizure medicines (e.g., levetiracetam) forseizure prophylaxis. Grade 3 Administer tocilizumab per Administerembodiment 15 for management dexamethasone of Grade 2 CRS. 10 mgintravenously In addition, administer dexamethasone every 6 hours. 10 mgintravenously with the first dose of Continue dexamethasone usetocilizumab and repeat dexamethasone until the event is Grade 1 or doseevery 6 hours. Continue less, then taper corticosteroids. dexamethasoneuse until the event is If not improving, manage as Grade 1 or less, thentaper Grade 4. corticosteroids. If improving, discontinue tocilizumaband manage as Grade 2. If still not improving, manage as Grade 4(below). Consider non-sedating anti-seizure medicines (e.g.,levetiracetam) for seizure prophylaxis. Grade 4 Administer tocilizumabper Administer embodiment 15 for management methylprednisolone 1000 mgof Grade 2 CRS. intravenously per day Administer methylprednisolone 1000mg for 3 days. intravenously per day with first dose of If improving,then manage as tocilizumab and continue Grade 3. methylprednisolone 1000mg If not improving, intravenously per day for 2 more days. consideralternate If improving, then manage as Grade 3. immunosuppressants Ifnot improving, consider alternate immunosuppressants. Considernon-sedating anti-seizure medicines (e.g., levetiracetam) for seizureprophylaxis.

Embodiment 25. The method of any one of embodiments 1 through 24,wherein the MCL subject is a high-risk patient as determined by a Ki-67tumor proliferation index ≥50% and/or presence of a TP53 mutation.

Embodiment 26. The method of any one of embodiments 20 through 22,wherein CRS in a B cell ALL subject is managed according to thefollowing protocol:

CRS Grade Tocilizumab Corticosteroids Grade 1 If not improving after Notapplicable. Symptoms require 24 hours, administer tocilizumabsymptomatic treatment only 8 mg/kg intravenously over (e.g., fever,nausea, fatigue, 1 hour (not to exceed 800 mg). headache, myalgia,malaise). Grade 2 Administer tocilizumab Manage per Grade 3 Symptomsrequire and 8 mg/kg intravenously over if no improvement respond tomoderate 1 hour (not to exceed 800 mg). within 24 hours afterintervention. Repeat tocilizumab every starting tocilizumab. Oxygenrequirement less than 8 hours as needed if not If improving, taper 40%FiO2 or hypotension responsive to intravenous fluids corticosteroids.responsive to fluids or low or increasing supplemental dose of onevasopressor or oxygen. Limit to a maximum of Grade 2 organ toxicity. 3doses in a 24-hour period; maximum total of 4 doses if no clinicalimprovement in the signs and symptoms of CRS. If improving, discontinuetocilizumab. Grade 3 Per Grade 2 Administer Symptoms require andmethylprednisolone respond to aggressive 1 mg/kg intravenouslyintervention. twice daily or Oxygen requirement greater equivalent thanor equal to 40% FiO2 or dexamethasone (e.g., hypotension requiring high-10 mg intravenously dose or multiple vasopressors every 6 hours) untilor Grade 3 organ toxicity or Grade 1, then taper Grade 4 transaminitis.corticosteroids. If improving, manage as Grade 2. If not improving,manage as Grade 4. Grade 4 Per Grade 2 Administer Life-threateningsymptoms. methylprednisolone Requirements for ventilator 1000 mg supportor continuous veno- intravenously per venous hemodialysis day for 3days. (CVVHD), or Grade 4 organ If improving, taper toxicity (excludingcorticosteroids, and transaminitis). manage as Grade 3. If notimproving, consider alternate immunosuppressants.

Embodiment 27. The method of any one of embodiments 20 through 22 and26, wherein neurologic toxicity in a B cell ALL subject is managed inaccordance with one of the following two protocols:

NE Revised Management Grade Original Management Guidelines GuidelinesGrade 1 Supportive care Supportive care Neurological examination andClosely monitor neurologic additional work-up as clinically statusindicated Consider prophylactic antiepileptic Grade 2 Supportive Careand Evaluation Supportive Care and Evaluation Neurological examination,brain MRI, Continuous cardiac telemetry and evaluation of CSF; considerEEG as and pulse oximetry as clinically indicated indicated Considerprophylactic antiepileptic Serial neurological examinations to includefundoscopy and Glasgow Coma Score, brain MRI, evaluation of CSF, EEG;consider neurology consult Administer antiepileptics for patients withseizures Tocilizumab Tocilizumab Consider tocilizumab 8 mg/kg IV overFor patients with 1 hour (not to exceed 800 mg) for patients concurrentCRS, administer with comorbid conditions (eg, grade tocilizumab 8 mg/kgIV over ≥2 CRS) 1 hour (not to exceed 800 mg); repeat every 4-6 hours asneeded if not responsive to IV fluids or increasing supplemental oxygen,for a maximum of 3 doses in 24 hours Discontinue tocilizumab if patientimproves Corticosteroids Corticosteroids N/A For patients withoutconcurrent CRS, administer dexamethasone 10 mg IV every 6 hours Forpatients with concurrent CRS, if no improvement within 24 hours afterstarting tocilizumab, administer dexamethasone 10 mg IV every 6 hoursTaper corticosteroids if patient improves Grade 3 Supportive Care andEvaluation Supportive Care and Evaluation Per grade 2 Manage inmonitored care or Monitor with continuous cardiac ICU telemetry andpulse oximetry Tocilizumab Tocilizumab Consider tocilizumab 8 mg/kg IVover Per grade 2 1 hour (not to exceed 800 mg); repeat Discontinuetocilizumab if every 4-6 hours if symptoms have not patient improvesstabilized or improved Corticosteroids Corticosteroids Considercorticosteroids (eg, Administer dexamethasone dexamethasone 10 mg IVevery 6 hours 10 mg IV every 6 hours or methylprednisolone l mg/kg BID)Taper corticosteroids if for worsening symptoms despite patient improvestocilizumab Grade 4 Supportive Care and Evaluation Supportive Care andEvaluation Per grade 2 Per grade 3 Monitor with continuous cardiacMechanical ventilation may telemetry and pulse oximetry be requiredAdminister immunosuppresants if patient does not improve TocilizumabTocilizumab Administer tocilizumab per grade 3 if Per grade 2 notpreviously administered Corticosteroids Corticosteroids Administercorticosteroids (eg, Administer high-dose methylprednisolone 1 g/d × 3days, corticosteroids (eg, followed by 250 mg BID × 2 days, thenmethylprednisone 1 g/d × 125 mg BID × 2 days, then 60 mg BID × 3 days) 2days) Taper corticosteroids if patient improves

Embodiment 28. The method of any one of embodiments 1 through 27,wherein the B cell ALL subject may receive any one or more of thefollowing bridging chemotherapy regimens:

Predefined Bridging Chemotherapy Regimens Attenuated VAD Vincristinenon-liposomal (1-2 mg IV weekly) or liposomal (2.25 mg/m² IV weekly),and dexamethasone 20-40 mg IV or PO daily × 3-4 days per week. Optionaldoxorubicin 50 mg/m² IV × 1 (first week only) Mercaptopurine 50-75mg/m²/day by mouth (administer at bedtime on an empty (6-MP) stomach toimprove absorption) Hydroxyurea Doses titrated between 15-50 mg/kg/day(rounded to the nearest 500 mg capsule and given as a single daily oraldose on a continuous basis) DOMP Dexamethasone 6 mg/m²/day PO (or IV)divided BID days 1-5, vincristine 1.5 mg/m² (maximum dose 2 mg) IV onday 1, methotrexate 20 mg/m² PO weekly, 6-MP 50-75 mg/m²/day PO dailyAttenuated Fludarabine 30 mg/m² IV days 1-2, cytarabine 2 g/m² IV days1-2, FLAG/FLAG-IDA G-CSF 5 μg/kg SC or IV starts on day 3 and cancontinue until day before the start of conditioning chemotherapy. Withor without idarubicin 6 mg/m² IV days 1-2 Mini-hyper CVAD Course A:Cyclophosphamide 150 mg/m² every 12 h × 3 days, (courses A and/ordexamethasone 20 mg/d IV or PO daily days 1-4 and 11-14, B) vincristine2 mg IV × 1 Course B: methotrexate 250 mg/m² IV over 24 hours on day 1,cytarabine 0.5 g/m² IV every 12 hours × 4 doses on days 2 and 3

Embodiment 29. Autologous T cells expressing an anti-CD19 CAR for use ina method for treating mantle cell lymphoma (MCL) or B cell ALL accordingto any one of embodiments 1 through 28.

Embodiment 30. Use of autologous T cells expressing an anti-CD19 CAR inthe manufacturing of a medicament for treating mantle cell lymphoma(MCL) or B cell ALL according to any one of embodiments 1 through 28.

Embodiment 31. A method of predicting:

-   -   (i) objective response of a subject to a CAR T cell treatment        (optionally, according to the method of any one of embodiments 1        through 28) comprising measuring peak CAR T cell levels and        comparing them to a reference standard, wherein objective        response is positively associated with peak CAR T cell levels,        wherein objective response includes both complete response and        partial response, and wherein all responses are assessed using        the Lugano classification.    -   (ii) minimal residual disease (e.g., at week 4) in response to a        CAR T cell treatment (optionally, according to the method of any        one of embodiments 1 through 28) comprising measuring peak CAR T        cell levels and comparing them to a reference standard, wherein        negative minimal residual disease is associated with higher peak        CAR T cell levels.    -   (iii) grade ≥3 CRS and/or grade ≥3 neurologic events (NE) in a        subject receiving CAR T cell treatment (optionally, according to        a method of any one of embodiments 1 through 28) comprising        measuring peak CAR T cell expansion after treatment and        comparing the levels to a reference value, wherein the higher        the CAR T cell expansion, the higher the chance for grade ≥3 CRS        and/or grade ≥3 NE events.    -   (iv) grade ≥3 CRS and/or grade ≥3 NE comprising measuring the        peak levels of GM-CSF and IL-6 post-CAR T cell treatment        (optionally, according to the method of any one of embodiments 1        through 28) and comparing them to a reference level, wherein the        higher the peak level of these cytokines, the higher the chance        for grade ≥3 CRS and/or grade ≥3 NE.    -   (v) grade ≥3 CRS in a subject receiving CAR T cell treatment        (optionally, according to a method of any one of embodiments 1        through 28) comprising measuring the peak level of serum        ferritin post-CAR T cell treatment and comparing it to a        reference level, wherein the higher the peak level of ferritin,        the higher the chance for grade ≥3 CRS.    -   (vi) grade ≥3 CRS comprising measuring the peak levels of serum        IL-2 and IFN-gamma post-CAR T cell treatment (optionally, of any        one of embodiments 1 through 28) and comparing them to a        reference level, wherein the higher the peak level of IL-2 and        IFN-gamma, the higher the chance for grade ≥3 NE.    -   (vii) grade ≥3 CRS comprising measuring the cerebrospinal fluid        levels of C-reactive protein, ferritin, IL-6, IL-8, and/or        vascular cell adhesion molecule (VCAM) post-CAR T cell treatment        (optionally, of any one of embodiments 1 through 28) and        comparing them to a reference level, wherein the higher the        cerebrospinal fluid levels of C-reactive protein, ferritin,        IL-6, IL-8, and/or vascular cell adhesion molecule (VCAM), the        higher the chance for grade ≥3 NE    -   (viii) grade ≥3 CRS post-CAR T cell treatment (optionally,        according to a method of any one of embodiments 1 through 28)        comprising measuring peak serum levels of IL-15, IL-2 Ra, IL-6,        TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A,        granzyme B, and/or perforin after anti-CD19 CAR T treatment and        comparing the levels to reference levels, wherein the peak serum        levels of IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10,        IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and/or perforin        associate positively with grade ≥3 CRS.    -   (ix) grade ≥3 CRS post-CAR T cell treatment of B cell ALL        (optionally, according to a method of any one of embodiments 1        through 28) comprising measuring peak serum level of IL-15 after        anti-CD19 CAR T treatment and comparing the levels to reference        levels, wherein the peak serum level of IL-15 associates        negatively with grade ≥3 CRS.    -   (x) grade ≥3 CRS and/or grade ≥3 NE post-CAR T cell treatment        (optionally, according to a method of any one of embodiments 1        through 28) comprising measuring peak serum levels of IL-6,        TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B after anti-CD19 CAR        T treatment and comparing the levels to reference levels,        wherein peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b,        and granzyme B associate positively with grade ≥3 CRS and grade        ≥3 NE.    -   (xi) whether a patient is going to be MRD (10⁻⁵ sensitivity)        negative at 4 weeks/one month post-CAR T cell treatment        (optionally, of any one of embodiments 1 through 28), comprising        measuring peak serum levels of IFN-γ, IL-6, and/or IL-2 after        treatment and comparing the level to a reference standard,        wherein peak serum levels of IFN-γ, IL-6, and/or IL-2 associate        positively with being MRD negative at one month.

Embodiment 32. The method of any one of embodiments 20 through 24, 26,27, and 30 through 31, wherein CRS and NE are graded by the methoddescribed in Lee et al., Blood 2014; 124: 188-195.

Embodiment 33. The method of embodiment 31, wherein the referencestandard is established by any method generally used in the biomarkerarts, such as quartile analysis of patient populations with knownresponses, grades of toxicity, and MRD levels.

Embodiment 34. The method of embodiment 31, wherein CAR T cell levelsare measured by CAR gene copies per microgram of DNA in blood.

Embodiment 35. The method of any one of embodiments 1 through 43,further comprising reducing the levels/activity of the cytokines thatassociate positively with grade ≥3 CRS and/or grade ≥3 NE post CAR Tcell infusion to reduce grade ≥3 CRS and/or grade ≥3 NE.

Embodiment 36. A method of improving the effectiveness of CAR T celltreatment (e.g., of classical, blastoid, and pleomorphic MCL, and B cellALL), in a subject in need thereof, comprising manipulating the T cellphenotype of the T cell product administered to the subject, optionallywherein the manipulation comprises increasing the number of CD3+ Tcells, decreasing the number of CD3− cells, increasing thenumber/percentage of CDRA45+ CCR7+(naïve-like) T cells and/or decreasingthe number/percentage of differentiated cells in the T cell productduring production, decreasing the levels of IFN-gamma production by theT cells, wherein the improvement is observed relative to theeffectiveness of a T cell product that is prepared without anyintentional manipulation of the number/percentage of CDRA45+CCR7+(naïve-like) T cells and/or the number/percentage of differentiatedcells in the T cell product.

BRIEF DESCRIPTIONS OF DRAWINGS

FIGS. 1A-1F: Comparable pharmacodynamic profile in prognostic groupsdefined by Ki-67 proliferation index, and trend for increased cytokinelevels in patients with mutated TP53.

FIGS. 2A-2I: Increased peak levels of select cytokines in serum amongpatients who achieved MRD-negative status.

FIG. 3 : ZUMA-3 Study Design. CAR, chimeric antigen receptor; DLT,dose-limiting toxicity.

FIG. 4 .: ZUMA-3 CONSORT Diagram. * AEs were grade 3 pulmonary mass(n=1), grade 1 subdural hematoma (n=1), and grade 3 febrile neutropenia(n=1); †AEs were grade 4 sepsis (n=1) and grade 5 sepsis (n=1); ‡Onepatient did receive KTE-X19 under compassionate use due to deep veinthrombosis, a study exclusion criterion. AE, adverse event.

FIG. 5 : Subgroup analysis of complete response rate. BM, bone marrow;ORR, overall remission rate; SCT, stem cell transplant.

FIG. 6 : Duration of response, relapse-free survival, and overallsurvival by dose level.

FIG. 7 : Peak CAR T-cell expansion and associations with response,minimal residual disease, and toxicity.

FIG. 8 : CAR T-cell area under the curve associations with response,minimal residual disease, and toxicity. AE, adverse event; AUC, areaunder the curve; CAR, chimeric antigen receptor; CRS, cytokine releasesyndrome; MRD, minimal residual disease.

FIG. 9 : Peak cytokine levels over time.

FIG. 10 : Inflammatory markers in blood serum samples at baseline and atpost-infusion peak. * Value represents lower limit of quantification inassay used. † Value represents upper limit of quantification in assayused. AE, adverse event; CAR, chimeric antigen receptor; CCL, C-C motifligand; CRP, C-reactive protein; CXCL, C-X-C motif chemokine ligand;FGFBF, fibroblast growth factor basic form; FLT-1, fms related receptortyrosine kinase 1; GM-CSF, granulocyte-macrophage colony-stimulatingfactor; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IL,interleukin; MCP, monocyte chemoattractant protein-1; MDC,macrophage-derived chemokine; MIP, macrophage inflammatory protein;PDL1, programmed death ligand 1; PLGF, placental growth factor; Ra,receptor alpha; RA, receptor antagonist; SAA, serum amyloid A; SFASL,soluble Fas ligand; TARC, thymus and activation-regulated cytokine; TNF,tumor necrosis factor; VCAM, vascular cell adhesion protein; VEGF,vascular endothelial growth factor; VEGFC, vascular endothelial growthfactor C; VEGFD, vascular endothelial growth factor D.

FIG. 11 : Association of Serum Biomarkers with Cytokine Release Syndromeand Neurologic Events. * Value represents lower limit of quantificationin assay used. † Value represents upper limit of quantification in assayused. CRP, C-reactive protein; CXCL, C-X-C motif chemokine ligand;GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN□,interferon gamma; IL, interleukin; IP, interferon γ-induced protein;MCP, monocyte attractant protein; Ra, receptor alpha; RA, receptorantagonist; SAA, serum amyloid A.

FIG. 12 : Pharmacodynamic profile of KTE-X19 across MCL morphologysubgroups. AUC, area under the curve; CAR, chimeric antigen receptor;CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon gamma; IL,interleukin; MCL, mantle cell lymphoma; MCP-1, monocyte chemoattractantprotein-1; MIP-1β, macrophage inflammatory protein-1 beta; PD-L1,programmed death-ligand 1; PRF, perforin; Rα, receptor alpha; TNF-α,tumor necrosis factor alpha.

FIG. 13 : Pharmacological profile of KTE-X19 across MCL morphologysubgroups.

FIG. 14 : Pharmacodynamic profile of KTE-X19 across prior BTKisubgroups. AUC, area under the curve; CAR, chimeric antigen receptor;CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon gamma; IL,interleukin; MCL, mantle cell lymphoma; MCP-1, monocyte chemoattractantprotein-1; MIP-1β, macrophage inflammatory protein-1 beta; PD-L1,programmed death-ligand 1; PRF, perforin; Rα, receptor alpha; TNF-α,tumor necrosis factor alpha.

FIG. 15 : Pharmacological profile of KTE-X19 across prior BTKisubgroups.

FIG. 16 : Ongoing response rate across subgroups.

DETAILED DESCRIPTION

Except as otherwise expressly provided herein, each of the followingterms shall have the meaning set forth below. Additional definitions areset forth throughout the application. Unless defined otherwise, alltechnical and scientific terms used herein have the meaning as commonlyunderstood by one of ordinary skill in the art. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary of Biochemistry andMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisapplication.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The disclosure provided herein are notlimitations of the various aspects of the application, which may be byreference to the specification as a whole. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure is related. For example, Juo, “The Concise Dictionary ofBiomedicine and Molecular Biology”, 2nd ed., (2001), CRC Press; “TheDictionary of Cell & Molecular Biology”, 5th ed., (2013), AcademicPress; and “The Oxford Dictionary Of Biochemistry And MolecularBiology”, Cammack et al. eds., 2nd ed, (2006), Oxford University Press,provide those of skill in the art with a general dictionary for many ofthe terms used in this disclosure.

The articles “a” or “an” refer to “one or more” of any recited orenumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for certain valueor composition as determined by one of ordinary skill in the art, whichwill depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” may mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” may mean a rangeof up to 10% (i.e., ±10%). For example, about 3 mg may include anynumber between 2.7 mg and 3.3 mg (for 10%). With respect to biologicalsystems or processes, the terms may mean up to an order of magnitude orup to 5-fold of a value. When certain values or compositions areprovided in the application and claims, unless otherwise stated, themeaning of “about” or “comprising essentially of” include an acceptableerror range for that value or composition. Any concentration range,percentage range, ratio range, or integer range includes the value ofany integer within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive and covers both “or” and “and”.The term “and/or” refer to each of the two specified features orcomponents with or without the other. Thus, the term “and/or” as used ina phrase such as “A and/or B” herein is intended to include “A and B,”“A or B,” “A” (alone), and “B” (alone). Similarly, the term “and/or” asused in a phrase such as “A, B, and/or C” is intended to encompass eachof the following aspects: A, B, and C; A, B, or C; A or C; A or B; B orC; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The terms “e.g.,” and “i.e.” are used merely by way of example, withoutlimitation intended, and not be construed as referring only those itemsexplicitly enumerated in the specification.

The terms “or more”, “at least”, “more than”, and the like, e.g., “atleast one” include but not be limited to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more than the statedvalue. Also included is any greater number or fraction in between. Theterm “no more than” includes each value less than the stated value. Forexample, “no more than 100 nucleotides” includes 100, 99, 98, 97, 96,95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78,77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60,59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, 1, and 0 nucleotides. Also included is any lesser number orfraction in between.

The terms “plurality”, “at least two”, “two or more”, “at least second”,and the like include but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also included isany greater number or fraction in between.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” is understood to imply the inclusion ofa stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps. It is understood that whereveraspects are described herein with the language “comprising,” otherwiseanalogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided. The term “consisting of”excludes any element, step, or ingredient not specified in the claim. Inre Gray, 53 F.2d 520, 11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ448, 450 (Bd. App. 1948) (“consisting of” defined as “closing the claimto the inclusion of materials other than those recited except forimpurities ordinarily associated therewith”). The term “consistingessentially of” limits the scope of a claim to the specified materialsor steps “and those that do not materially affect the basic and novelcharacteristic(s)” of the claimed invention.

Unless specifically stated or evident from context, as used herein, theterm “about” refers to a value or composition that is within anacceptable error range for the particular value or composition asdetermined by one of ordinary skill in the art, which will depend inpart on how the value or composition is measured or determined, i.e.,the limitations of the measurement system. For example, “about” or“approximately” may mean within one or more than one standard deviationper the practice in the art. “About” or “approximately” may mean a rangeof up to 10% (i.e., ±10%). Thus, “about” may be understood to be within10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or0.001% greater or less than the stated value. For example, about 5 mgmay include any amount between 4.5 mg and 5.5 mg. Furthermore,particularly with respect to biological systems or processes, the termsmay mean up to an order of magnitude or up to 5-fold of a value. Whenparticular values or compositions are provided in the instantdisclosure, unless otherwise stated, the meaning of “about” or“approximately” should be assumed to be within an acceptable error rangefor that particular value or composition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to be inclusive of the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

The term “activation,” “activated,” or the like refers to the state of acell, including and not be limited to an immune cell (e.g., a T cell),that has been sufficiently stimulated to induce detectable cellularproliferation. Activation may be associated with induced cytokineproduction and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision. T cell activation may be characterized by increased T cellexpression of one or more biomarker, including, but not limited to,CD57, PD1, CD107a, CD25, CD137, CD69, and/or CD71. Methods foractivating and expanding T cells are known in the art and are described,e.g., in U.S. Pat. Nos. 6,905,874; 6,867,041; and 6,797,514; and PCTPublication No. WO 2012/079000, the contents of which are herebyincorporated by reference in their entirety. In general, such methodsinclude contacting cells (such as T cells) with an activating,stimulatory, or costimulatory agent (such as anti-CD3 and/or anti-CD28antibodies) which may be attached, coated, or bound to a bead or othersurface, in a solution (such as feeding, culture, and/or growth medium)with certain cytokines (such as IL-2, IL-7, and/or IL-15). Theactivation agent (such as anti-CD3 and/or anti-CD28 antibodies) attachedto the same bead serve as a “surrogate” antigen presenting cell (APC).One example is The Dynabeads® system, a CD3/CD28 activator/stimulatorsystem for physiological activation of human T cells. In one embodiment,the T cells are activated and stimulated to proliferate with certainantibodies and/or cytokines using the methods described in U.S. Pat.Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012/129514, thecontents of which are hereby incorporated by reference in theirentirety.

The terms “administration,” “Administering” or the like refer tophysical introduction of an agent to a subject, using any of the variousmethods and delivery systems known to those skilled in the art.Exemplary routes of administration for the immune cells prepared by themethods disclosed herein include intravenous (i.v. or IV),intramuscular, subcutaneous, intraperitoneal, spinal or other parenteralroutes of administration, for example by injection or infusion.Parenteral route of administration refer to modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion, as well as in vivoelectroporation. In one embodiment, the immune cells (e.g., T cells)prepared by the present methods are administered via injection orinfusion. Non-parenteral routes include a topical, epidermal or mucosalroute of administration, for example, intranasally, vaginally, rectally,sublingually or topically. Administering may also be once, twice, or aplurality of times over one or more extended periods. Where one or moretherapeutic agents (e.g., cells) are administered, the administrationmay be done concomitantly or sequentially. Sequential administrationcomprises administration of one agent only after administration of theother agent or agents has been completed.

The term “antibody” (Ab) includes, without limitation, an immunoglobulinwhich binds specifically to an antigen. In general, an antibody maycomprise at least two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. Each H chain comprises a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region may comprise three or fourconstant domains, CH1, CH2 CH3, and/or CH4. Each light chain comprises alight chain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region may comprise oneconstant domain, CL. The VH and VL regions may be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each VH and VL comprises three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An immunoglobulinmay derive from any of the commonly known isotypes, including but notlimited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also wellknown to those in the art and include but are not limited to human IgG1,IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g.,IgM or IgG1) that is encoded by the heavy chain constant region genes.The term “antibody” includes, by way of example, both naturallyoccurring and non-naturally occurring Abs; monoclonal and polyclonalAbs; chimeric and humanized Abs; human or nonhuman Abs; wholly syntheticAbs; and single chain Abs. A nonhuman Ab may be humanized by recombinantmethods to reduce its immunogenicity in man Where not expressly stated,and unless the context indicates otherwise, the term “antibody” alsoincludes an antigen-binding fragment or an antigen-binding portion ofany of the aforementioned immunoglobulins, a monovalent and a divalentfragment or portion, and a single chain Ab.

An “antigen binding molecule,” “antibody fragment” or the like refer toany portion of an antibody less than the whole. An antigen bindingmolecule may include the antigenic complementarity determining regions(CDRs). Examples of antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)2, and Fv fragments, dAb, linear antibodies, scFvantibodies, and multispecific antibodies formed from antigen bindingmolecules. In one aspect, the CD19 CAR construct comprises an anti-CD 19single-chain FV. A “Single-chain Fv” or “scFv” antibody binding fragmentcomprises the variably heavy (V_(H)) and variable light (V_(L)) domainsof an antibody, where these domains are present in a single polypeptidechain. Generally, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains, which enables the scFv toform the desired structure for antigen binding. All antibody-relatedterms used herein take the customary meaning in the art and are wellunderstood by one of ordinary skill in the art.

An “antigen” refers to any molecule that provokes an immune response oris capable of being bound by an antibody or an antigen binding molecule.The immune response may involve either antibody production, or theactivation of specific immunologically-competent cells, or both. Aperson of skill in the art would readily understand that anymacromolecule, including virtually all proteins or peptides, may serveas an antigen. An antigen may be endogenously expressed, i.e. expressedby genomic DNA, or may be recombinantly expressed. An antigen may bespecific to a certain tissue, such as a cancer cell, or it may bebroadly expressed. In addition, fragments of larger molecules may act asantigens. In some embodiments, antigens are tumor antigens.

The term “neutralizing” refers to an antigen binding molecule, scFv,antibody, or a fragment thereof, that binds to a ligand and prevents orreduces the biological effect of that ligand. In some embodiments, theantigen binding molecule, scFv, antibody, or a fragment thereof,directly blocking a binding site on the ligand or otherwise alters theligand's ability to bind through indirect means (such as structural orenergetic alterations in the ligand). In some embodiments, the antigenbinding molecule, scFv, antibody, or a fragment thereof prevents theprotein to which it is bound from performing a biological function

The term “autologous” refers to any material derived from the sameindividual to which it is later to be re-introduced. For example, theengineered autologous cell therapy method described herein involves acollection of lymphocytes from an individual (such as a donor or apatient), which are then engineered to express a CAR construct and thenadministered back to the same individual.

The term “allogeneic” refers to any material derived from one individualwhich is then introduced to another individual of the same species,e.g., allogeneic T cell transplantation.

A “cancer” refers to a broad group of various diseases characterized bythe uncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth results in the formation of malignant tumors thatinvade neighboring tissues and may also metastasize to distant parts ofthe body through the lymphatic system or bloodstream. A “cancer” or“cancer tissue” may include a tumor at various stages. In oneembodiment, the cancer or tumor is stage 0, such that, e.g., the canceror tumor is very early in development and has not metastasized. Inanother embodiment, the cancer or tumor is stage I, such that, e.g., thecancer or tumor is relatively small in size, has not spread into nearbytissue, and has not metastasized. In other embodiment, the cancer ortumor is stage II or stage III, such that, e.g., the cancer or tumor islarger than in stage 0 or stage I, and it has grown into neighboringtissues but it has not metastasized, except potentially to the lymphnodes. In additional embodiment, the cancer or tumor is stage IV, suchthat, e.g., the cancer or tumor has metastasized. Stage IV may also bereferred to as advanced or metastatic cancer.

An “anti-tumor effect” as used herein, refers to a biological effectthat may present, and not being limited to, as a decrease in tumorvolume, an inhibition of tumor growth, a decrease in the number of tumorcells, a decrease in tumor cell proliferation, a decrease in thenumber/extent of metastases, an increase in overall or progression-freesurvival, an increase in life expectancy, and/or amelioration of variousphysiological symptoms associated with the tumor. An anti-tumor effectmay also refer to the prevention of the occurrence of a tumor, e.g., avaccine.

The term “progression-free survival” (PFS) refers to the time from thetreatment date to the date of disease progression (per generalguidelines, such as revised IWG Response Criteria for MalignantLymphoma) or death from any cause. The term “Disease progression” may beassessed by measurement of malignant lesions on radiographs or othermethods should not be reported as adverse events. Death due to diseaseprogression in the absence of signs and symptoms may be reported as theprimary tumor type (e.g., DLBCL). The term “duration of response” (DOR)refers to the period of time between a subject's first objectiveresponse to the date of confirmed disease progression (per generalguidelines, such as the revised IWG Response Criteria for MalignantLymphoma) or death. The term “overall survival” (OS) refers to the timefrom the date of treatment to the date of death.

A “cytokine” refers to a non-antibody protein that may be released byimmune cells, including macrophages, B cells, T cells, and mast cells topropagate an immune response. In one embodiment, one or more cytokinesare released in response to the therapy. In other embodiment, thosecytokines secreted in response to the therapy may indicate or suggest aneffective therapy. In one embodiment, “cytokine” refers to anon-antibody protein that is released by one cell in response to contactwith a specific antigen, wherein the cytokine interacts with a secondcell to mediate a response in the second cell. “Cytokine” as used hereinis meant to refer to proteins released by one cell population that acton another cell as intercellular mediators. A cytokine may beendogenously expressed by a cell or administered to a subject. Cytokinesmay be released by immune cells, including macrophages, B cells, Tcells, and mast cells to propagate an immune response. Cytokines mayinduce various responses in the recipient cell. Cytokines may includehomeostatic cytokines, chemokines, pro-inflammatory cytokines,effectors, and acute-phase proteins. For example, homeostatic cytokines,including interleukin (IL) 7 and IL-15, promote immune cell survival andproliferation, and pro-inflammatory cytokines may promote aninflammatory response. Examples of homeostatic cytokines include, butare not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70,IL-15, and interferon (IFN) gamma Examples of pro-inflammatory cytokinesinclude, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a,tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor(FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF),soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascularadhesion molecule 1 (sVCAM-1), vascular endothelial growth factor(VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples ofeffectors include, but are not limited to, granzyme A, granzyme B,soluble Fas ligand (sFasL), and perforin. Examples of acutephase-proteins include, but are not limited to, C-reactive protein (CRP)and serum amyloid A (SAA).

“Chemokines” are a type of cytokine that mediates cell chemotaxis, ordirectional movement. Examples of chemokines include, but are notlimited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derivedchemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 orCCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a),MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymus andactivation regulated chemokine (TARC or CCL17).

A “therapeutically effective amount,” “therapeutically effectivedosage,” or the like refers to an amount of the cells (such as immunecells or engineered T cells) that are produced by the present methods(resulting in a T cell product) and that, when used alone or incombination with another therapeutic agent, protects or treats a subjectagainst the onset of a disease or promotes disease regression asevidenced by a decrease in severity of disease symptoms, an increase infrequency and duration of disease symptom-free periods, and/orprevention of impairment or disability due to disease affliction. Theability to promote disease regression may be evaluated using a varietyof methods known to the skilled practitioner, such as in subjects duringclinical trials, in animal model systems predictive of efficacy inhumans, or by assaying the activity of the agent in in vitro assays. Insome embodiments, the donor T cells for use in the T cell therapy areobtained from the patient (e.g., for an autologous T cell therapy). Inother embodiments, the donor T cells for use in the T cell therapy areobtained from a subject that is not the patient. The T cells may beadministered at a therapeutically effective amount. For example, atherapeutically effective amount of the T cells may be at least about10⁴ cells, at least about 10⁵ cells, at least about 10⁶ cells, at leastabout 10⁷ cells, at least about 10⁸ cells, at least about 10⁹, or atleast about 10¹⁰. In another embodiment, the therapeutically effectiveamount of the T cells is about 10⁴ cells, about 10⁵ cells, about 10⁶cells, about 10⁷ cells, or about 10⁸ cells. In some embodiments, thetherapeutically effective amount of the CAR T cells is about 2×10⁶cells/kg, about 3×10⁶ cells/kg, about 4×10⁶ cells/kg, about 5×10⁶cells/kg, about 6×10⁶ cells/kg, about 7×10⁶ cells/kg, about 8×10⁶cells/kg, about 9×10⁶ cells/kg, about 1×10⁷ cells/kg, about 2×10⁷cells/kg, about 3×10⁷ cells/kg, about 4×10⁷ cells/kg, about 5×10⁷cells/kg, about 6×10⁷ cells/kg, about 7×10⁷ cells/kg, about 8×10⁷cells/kg, or about 9×10⁷ cells/kg. In some embodiments, thetherapeutically effective amount of the CAR-positive viable T cells isbetween about 1×10⁶ and about 2×10⁶ CAR-positive viable T cells per kgbody weight up to a maximum dose of about 1×10⁸ CAR-positive viable Tcells. In some embodiments, the therapeutically effective amount of theCAR-positive viable T cells is between about 0.4×10⁸ and about 2×10⁸CAR-positive viable T cells. In some embodiments, the therapeuticallyeffective amount of the CAR-positive viable T cells is about 0.4×10⁸,about 0.5×10⁸, about 0.6×10⁸, about 0.7×10⁸, about 0.8×10⁸, about0.9×10⁸, about 1.0×10⁸, about 1.1×10⁸, about 1.2×10⁸, about 1.3×10⁸,about 1.4×10⁸, about 1.5×10⁸, about 1.6×10⁸, about 1.7×10⁸, about1.8×10⁸, about 1.9×10⁸, or about 2.0×10⁸ CAR-positive viable T cells

The term “lymphocyte” as used herein may include natural killer (NK)cells, T cells, NK-T cells, or B cells. NK cells are a type of cytotoxic(cell toxic) lymphocyte that represent a major component of the inherentimmune system. NK cells reject tumors and cells infected by viruses,through the process of apoptosis or programmed cell death. They weretermed “natural killers” because they do not require activation to killcells. T-cells play a major role in cell-mediated immunity (no antibodyinvolvement). The T-cell receptors (TCR) differentiate themselves fromother lymphocyte types. The thymus, a specialized organ of the immunesystem, is primarily responsible for the T cell's maturation.

There are several types of “immune cells,” including, withoutlimitation, macrophages (e.g, tumor associated macrophages) neutrophils,basophils, eosinophils, granulocytes, natural killer cells (NK cells), Bcells, T cells, NK-T cells, mast cells, tumor infiltrating lymphocytes(TILs), myeloid derived suppressor cells (MDSCs), and dendritic cells.The term also includes precursors of these immune cells. Hematopoieticstem and/or progenitor cells may be derived from bone marrow, umbilicalcord blood, adult peripheral blood after cytokine mobilization, and thelike, by methods known in the art. Some precursor cells are those thatmay differentiate into the lymphoid lineage, for example, hematopoieticstem cells or progenitor cells of the lymphoid lineage. Additionalexamples of immune cells that may be used for immune therapy aredescribed in US Publication No. 20180273601, incorporated herein byreference in its entirety.

There are also several types of T-cells, namely: Helper T-cells (e.g.,CD4+ cells, effector TEFF cells), Cytotoxic T-cells (also known as TC,cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+T-cells or killer T cell), Memory T-cells ((i) stem memory T_(SCM)cells, like naive cells, are CD45RO−, CCR7+, CD45RA+,CD62L+(L-selectin), CD27+, CD28+ and IL-7Ra+, but they also expresslarge amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and show numerousfunctional attributes distinctive of memory cells); (ii) central memoryT_(CM) cells express L-selectin and are CCR7⁺ and CD45RO⁺ and theysecrete IL-2, but not IFNγ or IL-4, and (iii) effector memory TEM cells,however, do not express L-selectin or CCR7 but do express CD45RO andproduce effector cytokines like IFNγ and IL-4), Regulatory T-cells(Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), NaturalKiller T-cells (NKT), and Gamma Delta T-cells. T cells found withintumors are referred to as “tumor infiltrating lymphocytes” (TIL).B-cells, on the other hand, play a principal role in humoral immunity(with antibody involvement). It makes antibodies and antigens andperforms the role of antigen-presenting cells (APCs) and turns intomemory B-cells after activation by antigen interaction. In mammals,immature B-cells are formed in the bone marrow, where its name isderived from.

A “naïve” T cell refers to a mature T cell that remains immunologicallyundifferentiated. Following positive and negative selection in thethymus, T cells emerge as either CD4⁺ or CD8⁺ naïve T cells. In theirnaïve state, T cells express L-selectin (CD62L⁺), IL-7 receptor-α(IL-7R-α), and CD132, but they do not express CD25, CD44, CD69, orCD45RO. As used herein, “immature” may also refers to a T cell whichexhibits a phenotype characteristic of either a naïve T cell or animmature T cell, such as a T_(SCM) cell or a T_(CM) cell. For example,an immature T cell may express one or more of L-selectin (CD62L⁺),IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3,and LFA-1. Naïve or immature T cells may be contrasted with terminaldifferentiated effector T cells, such as TEM cells and TEE cells.

“T cell function,” as referred to herein, refers to normalcharacteristics of healthy T cells. T cell function may comprise T cellproliferation, T cell activity, and/or cytolytic activity. In oneembodiment, the methods of the present application of preparing T cellsunder certain oxygen and/or pressure condition would increase one ormore T cell function, thereby making the T cells more fit and/or morepotent for therapeutic purpose. In some embodiment, T cells that areprepared according to the present methods have increased T cell functionas compared to those under conditions lacking certain oxygen and/orpressure. In other embodiment, T cells that are prepared according tothe present methods would have increased T cell proliferation ascompared to T cells cultured under conditions lacking certain oxygenand/or pressure. In additional embodiment, T cells that are preparedaccording to the present methods have increased T cell activity ascompared to T cells cultured under conditions lacking certain oxygenand/or pressure. In further embodiment, T cells that are preparedaccording to the present methods have increased cytolytic activity ascompared to T cells cultured under conditions lacking certain oxygenand/or pressure.

The terms cell “proliferation,” “proliferating” or the like refer to theability of cells to grow in numbers through cell division. Proliferationmay be measured by staining cells with carboxyfluorescein succinimidylester (CFSE). Cell proliferation may occur in vitro, e.g., during T cellculture, or in vivo, e.g., following administration of a immune celltherapy (e.g., T cell therapy). The cell proliferation may be measuredor determined by the methods described herein or known in the field. Forexample, cell proliferation may be measured or determined by viable celldensity (VCD) or total viable cell (TVC). VCD or TVC may be theoretical(an aliquot or sample is removed from a culture at certain timepoint todetermine the cell number, then the cell number multiples with theculture volume at the beginning of the study) or actual (an aliquot orsample is removed from a culture at certain timepoint to determine thecell number, then the cell number multiples with the actual culturevolume at the certain timepoint). The term “T cell activity” refers toany activity common to healthy T cells. In one embodiment, the T cellactivity comprises cytokine production (such as INFγ, IL-2, and/orTNFα). In other embodiment, the T cell activity comprises production ofone or more cytokine selected from interferon gamma (IFNγ or IFN-γ),tissue necrosis factor alpha (TNFα or IFNα), and both. The terms“cytolytic activity,” “cytotoxicity” or the like refer to the ability ofa T cell to destroy a target cell. In one embodiment, the target cell isa cancer cell, e.g., a tumor cell. In other embodiment, the T cellexpresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR),and the target cell expresses a target antigen.

The term “genetically engineered,” “gene editing,” or “engineered”refers to a method of modifying the genome of a cell, including, but notbeing limited to, deleting a coding or non-coding region or a portionthereof or inserting a coding region or a portion thereof. In oneembodiment, the cell that is modified is a lymphocyte, e.g., a T cell,which may either be obtained from a patient or a donor. The cell may bemodified to express an exogenous construct, such as, e.g., a chimericantigen receptor (CAR) or a T cell receptor (TCR), which is incorporatedinto the cell's genome.

The terms “transduction” and “transduced” refer to the process wherebyforeign DNA is introduced into a cell via viral vector (see Jones etal., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ.(1998)). In some embodiments, the vector is a retroviral vector, a DNAvector, a RNA vector, an adenoviral vector, a baculoviral vector, anEpstein Barr viral vector, a papovaviral vector, a vaccinia viralvector, a herpes simplex viral vector, an adenovirus associated vector,a lentiviral vector, or any combination thereof

“Chimeric antigen receptors” (CARs or CAR-Ts) and the T cell receptors(TCRs) of the application are genetically engineered receptors. Theseengineered receptors may be readily inserted into and expressed byimmune cells, including T cells, in accordance with techniques known inthe art. With a CAR, a single receptor may be programmed to bothrecognize a specific antigen and, when bound to that antigen, activatethe immune cell to attack and destroy the cell bearing or expressingthat antigen. When these antigens exist on tumor cells, an immune cellthat expresses the CAR may target and kill the tumor cell. In oneembodiment, the cell that are prepared according to the presentapplication is a cell having a chimeric antigen receptor (CAR), or a Tcell receptor, comprising an antigen binding molecule, a costimulatorydomain, and an activating domain. The costimulatory domain may comprisean extracellular domain, a transmembrane domain, and an intracellulardomain. In one embodiment, the extracellular domain comprises a hinge ora truncated hinge domain.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including Abs, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom a vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues.

The terms “immunotherapy” “immune therapy” or the like refer to thetreatment of a subject afflicted with, or at risk of contracting orsuffering a recurrence of, a disease by a method comprising inducing,enhancing, suppressing or otherwise modifying an immune response.Examples of immunotherapy include, but are not limited to, T cell and NKcell therapies. T cell therapy may include adoptive T cell therapy,tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous celltherapy, engineered autologous cell therapy and allogeneic T celltransplantation. One of skill in the art would recognize that themethods of preparing immune cells disclosed herein would enhance theeffectiveness of any cancer or transplanted T cell therapy. Examples ofT cell therapies are described in U.S. Patent Publication Nos.2014/0154228 and 2002/0006409; U.S. Pat. Nos. 7,741,465; 6,319,494; and5,728,388; and PCT Publication No. WO 2008/081035, which areincorporated by reference in their entirety.

The term “engineered Autologous Cell Therapy,” which may be abbreviatedas “eACT™,” also known as adoptive cell transfer, is a process by whicha patient's own T cells are collected and subsequently geneticallyaltered to recognize and target one or more antigens expressed on thecell surface of one or more specific tumor cells or malignancies. Tcells may be engineered to express, for example, chimeric antigenreceptors (CAR) or T cell receptor (TCR). CAR positive (+) T cells areengineered to express an extracellular single chain variable fragment(scFv) with specificity for certain tumor antigen linked to anintracellular signaling part comprising a costimulatory domain and anactivating domain. The costimulatory domain may be a signaling regionderived from, e.g., CD28, CTLA4, CD16, OX-40, 4-1BB/CD137, CD2, CD7,CD27, CD30, CD40, programmed death-1 (PD-1), programmed death ligand-1(PD-L1), inducible T cell costimulator (ICOS), ICOS-L, lymphocytefunction-associated antigen-1 (LFA-1 (CD1 1a/CD18), CD3 gamma, CD3delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factorsuperfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fcgamma receptor, MHC class I molecule, TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), activating NK cellreceptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1,GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8, CD8alpha, CD8beta, IL2R beta, IL2R gamma,IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX,CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand thatspecifically binds with CD83, or any combination thereof. The activatingdomain may be derived from, e.g., CD3, such as CD3 zeta, epsilon, delta,gamma, or the like. In one embodiment, the CAR is designed to have two,three, four, or more costimulatory domains. The CAR scFv may be designedto target, for example, CD19, which is a transmembrane protein expressedby cells in the B cell lineage, including all normal B cells and B cellmalignances, including but not limited to NHL, CLL, and non-T cell ALL.Example CAR+ T cell therapies and constructs are described in U.S.Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and2014/0050708, which are hereby incorporated by reference in theirentirety.

A “costimulatory signal,” as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to aT cell response, such as, but not limited to, proliferation and/orupregulation or down regulation of key molecules.

A “costimulatory ligand,” as used herein, includes a molecule on anantigen presenting cell that specifically binds a cognate co-stimulatorymolecule on a T cell. Binding of the costimulatory ligand provides asignal that mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. Acostimulatory ligand induces a signal that is in addition to the primarysignal provided by a stimulatory molecule, for instance, by binding of aT cell receptor (TCR)/CD3 complex with a major histocompatibilitycomplex (MHC) molecule loaded with peptide. A co-stimulatory ligand mayinclude, but is not limited to, 3/TR6, 4-1BB ligand, agonist or antibodythat binds Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand,CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), humanleukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), ligand that specifically binds with B7-H3, lymphotoxinbeta receptor, MHC class I chain-related protein A (MICA), MHC class Ichain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death(PD) L1. A co-stimulatory ligand includes, without limitation, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28,CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83,lymphocyte function-associated antigen-1 (LFA-1), natural killer cellreceptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamilymember 14 (TNFSF14 or LIGHT).

A “costimulatory molecule” is a cognate binding partner on a T cell thatspecifically binds with a costimulatory ligand, thereby mediating acostimulatory response by the T cell, such as, but not limited to,proliferation. Costimulatory molecules include, but are not limited to,A “costimulatory molecule” is a cognate binding partner on a T cell thatspecifically binds with a costimulatory ligand, thereby mediating acostimulatory response by the T cell, such as, but not limited to,proliferation. Costimulatory molecules include, but are not limited to,4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19,CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha;beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a,CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86,CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1-1a, CD1-1b, CD1-1c, CD1-1d,CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS,GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2Rbeta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD,ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1,LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14;TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1(LFA-1 (CD1 1a/CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44,NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162),signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3),SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr,TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments,truncations, or combinations thereof.

In some aspect, the cells of the present application may be obtainedthrough T cells obtained from a subject. In one aspect, the T cells maybe obtained from, e.g., peripheral blood mononuclear cells (PBMC), bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Inaddition, the T cells may be derived from one or more T cell linesavailable in the art. T cells may also be obtained from a unit of bloodcollected from a subject using any number of techniques known to theskilled artisan, such as FICOLL™ separation and/or apheresis. In someaspect, the cells collected by apheresis are washed to remove the plasmafraction and placed in an appropriate buffer or media for subsequentprocessing. In some aspect, the cells are washed with any solution (e.g.a solution with neutralized PH value or PBS) or culture medium. As willbe appreciated, a washing step may be used, such as by using asemiautomated flow through centrifuge, e.g., the Cobe™ 2991 cellprocessor, the Baxter CytoMate™, or the like. In some aspect, the washedcells are resuspended in one or more biocompatible buffers, or othersaline solution with or without buffer. In some aspect, the undesiredcomponents of the apheresis sample are removed. Additional methods ofisolating T cells for a T cell therapy are disclosed in U.S. Patent Pub.No. 2013/0287748, which are hereby incorporated by references in theirentirety.

In some embodiments, T cells are isolated from PBMCs by lysing the redblood cells and depleting the monocytes, e.g., by using centrifugationthrough a PERCOLL™ gradient. In some embodiments, a specificsubpopulation of T cells, such as CD4+, CD8+, CD28+, CD45RA+, andCD45RO+ T cells is further isolated by positive or negative selectiontechniques known in the art. For example, enrichment of a T cellpopulation by negative selection may be accomplished with a combinationof antibodies directed to surface markers unique to the negativelyselected cells. In some embodiments, cell sorting and/or selection vianegative magnetic immunoadherence or flow cytometry that uses a cocktailof monoclonal antibodies directed to cell surface markers present on thecells negatively selected may be used. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typicallyincludes antibodies to CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In someembodiments, flow cytometry and cell sorting are used to isolate cellpopulations of interest for use in the present disclosure.

In one embodiment, CD3+ T cells are isolated from PBMCs using Dynabeadscoated with anti-CD3 antibody. CD8+ and CD4+ T cells are furtherseparately isolated by positive selection using CD8 microbeads (e.g.,Miltenyi Biotec) or CD4 microbeads (e.g., Miltenyi Biotec).

In some embodiments, PBMCs are used directly for genetic modificationwith the immune cells (such as CARs) using methods as described herein.In some embodiments, after isolating the PBMCs, T lymphocytes arefurther isolated, and both cytotoxic and helper T lymphocytes are sortedinto naive, memory, and effector T cell subpopulations either before orafter genetic modification and/or expansion

The one or more immune cells described herein may be obtained from anysource, including, for example, a human donor. The donor may be asubject in need of an anti-cancer treatment, e.g., treatment with oneimmune cells generated by the methods described herein (i.e., anautologous donor), or may be an individual that donates a lymphocytesample that, upon generation of the population of cells generated by themethods described herein, will be used to treat a different individualor cancer patient (i.e., an allogeneic donor) immune cells may bedifferentiated in vitro from a hematopoietic stem cell population, orimmune cells may be obtained from a donor. The population of immunecells may be obtained from the donor by any suitable method used in theart. For example, the population of lymphocytes may be obtained by anysuitable extracorporeal method, venipuncture, or other blood collectionmethod by which a sample of blood with or without lymphocytes isobtained. The population of lymphocytes is obtained by apheresis. Theone or more immune cells may be collected from any tissue that comprisesone or more immune cells, including, but not limited to, a tumor. Atumor or a portion thereof is collected from a subject, and one or moreimmune cells are isolated from the tumor tissue. Any T cell may be usedin the methods disclosed herein, including any immune cells suitable fora T cell therapy. For example, the one or more cells useful for theapplication may be selected from the group consisting of tumorinfiltrating lymphocytes (TIL), cytotoxic T cells, CAR T cells,engineered TCR T cells, natural killer T cells, Dendritic cells, andperipheral blood lymphocytes. T cells may be obtained from, e.g.,peripheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In addition, the T cells may bederived from one or more T cell lines available in the art. T cells mayalso be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as FICOLL™separation and/or apheresis. T cells may also be obtained from anartificial thymic organoid (ATO) cell culture system, which replicatesthe human thymic environment to support efficient ex vivodifferentiation of T-cells from primary and reprogrammed pluripotentstem cells. Additional methods of isolating T cells for a T cell therapyare disclosed in U.S. Patent Publication No. 2013/0287748, in PCTPublication Nos. WO2015/120096 and WO2017/070395, all of which areherein incorporated by reference in their totality for the purposes ofdescribing these methods and in their entirety. In one embodiment, Tcells are tumor infiltrating leukocytes. In certain embodiment, the oneor more T cells express CD8, e.g., are CD8⁺ T cells. In otherembodiment, the one or more T cells express CD4, e.g., are CD4⁺ T cells.Additional methods of isolating T cells for a T cell therapy aredisclosed in U.S. Patent Publication No. 2013/0287748, in PCTPublication Nos. WO2015/120096 and WO2017/070395, all of which areherein incorporated by reference in their totality for the purposes ofdescribing these methods and in their entirety.

The immune cells and their precursor cells may be isolated by availablemethods (see, for example, Rowland-Jones et al., Lymphocytes: APractical Approach, Oxford University Press, New York (1999)). Thesources for the immune cells or precursor cells thereof include, but arenot limited to, peripheral blood, umbilical cord blood, bone marrow, orother sources of hematopoietic cells. Negative selection methods may beused to remove cells that are not the desired immune cells.Additionally, positive selection methods may isolate or enrich fordesired immune cells or precursor cells thereof, or a combination ofpositive and negative selection methods may be employed. Monoclonalantibodies (MAbs) are useful for identifying markers associated withcertain cell lineages and/or stages of differentiation for both positiveand negative selections. If certain type of cell is to be isolated, forexample, certain type of T cell, various cell surface markers orcombinations of markers, including but not limited to, CD3, CD4, CD8,CD34 (for hematopoietic stem and progenitor cells) and the like, may beused to separate the cells, as is well known in the art (see Kearse, TCell Protocols: Development and Activation, Humana Press, Totowa N.J.(2000); De Libero, T Cell Protocols, Vol. 514 of Methods in MolecularBiology, Humana Press, Totowa N.J. (2009))

PBMCs may be used directly for genetic modification with the immunecells (such as CARs). After isolating the PBMCs, T lymphocytes arefurther isolated, and both cytotoxic and helper T lymphocytes are sortedinto naive, memory, and effector T cell subpopulations either before orafter genetic modification and/or expansion. In one embodiment, CD8+cells may be further sorted into naive, central memory, and effectorcells by identifying cell surface antigens that are associated with eachof these types of CD8+ cells. In other embodiment, the expression ofphenotypic markers of central memory T cells includes CCR7, CD3, CD28,CD45RO, CD62L, and CD127 and are negative for granzyme B. In someembodiment, central memory T cells are CD8+, CD45RO+, and CD62L+ Tcells. In certain embodiment, effector T cells are negative for CCR7,CD28, CD62L, and CD127 and positive for granzyme B and perforin. Inadditional embodiment, CD4+ T cells may be further sorted intosubpopulations. For example, CD4+T helper cells may be sorted intonaive, central memory, and effector cells by identifying cellpopulations that have cell surface antigens.

The methods described herein further comprise enriching or preparing apopulation of immune cells obtained from a donor, between harvestingfrom the donor and exposing one or more cells obtained from a donorsubject. Enrichment of a population of immune cells, e.g., the one ormore T cells, may be accomplished by any suitable separation methodincluding, but not limited to, the use of a separation medium (e.g.,FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte enrichment cocktail,Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No. 0850494X), orthe like), cell size, shape or density separation by filtration orelutriation, immunomagnetic separation (e.g., magnetic-activated cellsorting system, MACS), fluorescent separation (e.g., fluorescenceactivated cell sorting system, FACS), or bead-based column separation.

In one embodiment, the T cells are obtained from a donor subject. Inother embodiment, the donor subject is human patient afflicted with acancer or a tumor. In additional embodiment, the donor subject is ahuman patient not afflicted with a cancer or a tumor. The presentapplication also provides a composition or formulation comprises apharmaceutically acceptable carrier, diluent, solubilizer, emulsifier,preservative and/or adjuvant. In certain embodiment, the composition orformulation comprises an excipient. The terms composition andformulation are used interchangeably herein. The terms composition, atherapeutic composition, a therapeutically effective composition,pharmaceutical composition, pharmaceutically effective composition, anda pharmaceutically acceptable composition are used interchangeablyherein. The composition may be selected for parenteral delivery,inhalation, or delivery through the digestive tract, such as orally. Thecomposition may be prepared by known methods by one skilled person inthe art. Buffers are used to maintain the composition at physiologicalpH or at a slightly lower pH, typically within a pH range of from about5 to about 8. When parenteral administration is contemplated, thecomposition is in the form of a pyrogen-free, parenterally acceptableaqueous solution comprising a composition described herein, with orwithout additional therapeutic agents, in a pharmaceutically acceptablevehicle. By way of example, the vehicle for parenteral injection issterile distilled water in which composition described herein, with orwithout at least one additional therapeutic agent, is formulated as asterile, isotonic solution, properly preserved. The preparation involvesthe formulation of the desired agent with polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that providefor the controlled or sustained release of the product, which are thenbe delivered via a depot injection. In addition, implantable drugdelivery devices may be used to introduce the desired therapeutic agent.

In some embodiments, the donor T cells for use in the T cell therapy areobtained from the patient (e.g., for an autologous T cell therapy). Inother embodiments, the donor T cells for use in the T cell therapy areobtained from a subject that is not the patient. The T cells may beadministered at a therapeutically effective amount. For example, atherapeutically effective amount of the T cells may be at least about10⁴ cells, at least about 10⁵ cells, at least about 10⁶ cells, at leastabout 10⁷ cells, at least about 10⁸ cells, at least about 10⁹, or atleast about 10¹⁰. In another embodiment, the therapeutically effectiveamount of the T cells is about 10⁴ cells, about 10⁵ cells, about 10⁶cells, about 10⁷ cells, or about 10⁸ cells. In some embodiments, thetherapeutically effective amount of the CAR T cells is about 2×10⁶cells/kg, about 3×10⁶ cells/kg, about 4×10⁶ cells/kg, about 5×10⁶cells/kg, about 6×10⁶ cells/kg, about 7×10⁶ cells/kg, about 8×10⁶cells/kg, about 9×10⁶ cells/kg, about 1×10⁷ cells/kg, about 2×10⁷cells/kg, about 3×10⁷ cells/kg, about 4×10⁷ cells/kg, about 5×10⁷cells/kg, about 6×10⁷ cells/kg, about 7×10⁷ cells/kg, about 8×10⁷cells/kg, or about 9×10⁷ cells/kg. In some embodiments, thetherapeutically effective amount of the CAR-positive viable T cells isbetween about 1×10⁶ and about 2×10⁶ CAR-positive viable T cells per kgbody weight up to a maximum dose of about 1×10⁸ CAR-positive viable Tcells

A “patient” as used herein includes any human who is afflicted with adisease or disorder, including cancer (e.g., a lymphoma or a leukemia).The terms “subject” and “patient” are used interchangeably herein. Theterm “donor subject” refers to herein a subject whose cells are beingobtained for further in vitro engineering. The donor subject may be acancer patient that is to be treated with a population of cellsgenerated by the methods described herein (i.e., an autologous donor),or may be an individual who donates a lymphocyte sample that, upongeneration of the population of cells generated by the methods describedherein, will be used to treat a different individual or cancer patient(i.e., an allogeneic donor). Those subjects who receive the cells thatwere prepared by the present methods may be referred to as “recipientsubject.”

The terms “stimulation,” “stimulating,” or the like refer to a primaryresponse induced by binding of a stimulatory molecule with its cognateligand, wherein the binding mediates a signal transduction event. A“stimulatory molecule” is a molecule on a T cell, e.g., the T cellreceptor (TCR)/CD3 complex, that specifically binds with a cognatestimulatory ligand present on an antigen present cell. A “stimulatoryligand” is a ligand that when present on an antigen presenting cell(e.g., an artificial antigen presenting cell (aAPC), a dendritic cell, aB-cell, and the like) may specifically bind with a stimulatory moleculeon a T cell, thereby mediating a primary response by the T cell,including, but not limited to, activation, initiation of an immuneresponse, proliferation, and the like. Stimulatory ligands include, butare not limited to, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody. An “activated” or “active,” as used herein, refers toa T cell that has been stimulated. An active T cell may be characterizedby expression of one or more marker selected form CD137, CD25, CD71,CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

The term “exogenous activation materials” refers to any activationsubstance derived from an external source. For example, exogenousanti-CD3 antibody, anti-CD28 antibody, IL-2, exogenous IL-7, orexogenous IL-15 may be obtained commercially or produced recombinantly.“Exogenous IL-2,” “Exogenous IL-7,” or “exogenous IL-15” when added inor contacted with one or more T cells, indicates that such IL-2, IL-7and/or IL-15 are not produced by the T cells. The T cells prior to beingmixed with “Exogenous” IL-2, IL-7 or IL-15 may contain a trace amountthat were produced by the T cells or isolated from the subject with theT cells (i.e., endogenous “Exogenous” IL-2, IL-7 or IL-15). The one ormore T cells described herein may be contacted with exogenous anti-CD3antibody, anti-CD28 antibody, “Exogenous” IL-2, IL-7 and/or IL-15through any means known in the art, including addition of isolated“Exogenous” IL-2, IL-7 and/or IL-15 to the culture, inclusion ofanti-CD3 antibody, anti-CD28 antibody, “Exogenous” IL-2, IL-7 and/orIL-15 in the culture medium, or expression of “Exogenous” IL-2, IL-7and/or IL-15 by one or more cells in the culture other than the one ormore T cells, such as by a feeder layer.

As used herein, the term “in vitro cell” refers to any cell which iscultured ex vivo. In one embodiment, an in vitro cell includes a T cell.

The term “persistence” refers to the ability of, e.g., one or moretransplanted immune cells administered to a subject or their progenies(e.g., differentiated or matured T cells) to remain in the subject at adetectable level for a period of time. As used herein, increasing thepersistence of one or more transplanted immune cells or their progenies(e.g., differentiated or matured T cells) refers to increasing theamount of time the transplanted immune cells are detectable in a subjectafter administration. For example, the in vivo persistence of one ormore transplanted immune cells may be increased by at least about atleast about 1 day, at least about 2 days, at least about 3 days, atleast about 4 days, at least about 5 days, at least about 6 days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 3 weeks, atleast about 4 weeks, at least about 1 month, at least about 2 months, atleast about 3 months, at least about 4 months, at least about 5 months,or at least about 6 months. In addition, the in vivo persistence of oneor more transplanted immune cells may be increased by at least about1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about3-fold, at least about 3.5-fold, at least about 4-fold, at least about4.5-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, or at least about10-fold compared to the one or more transplanted immune cells that werenot prepared by the present methods disclosed herein.

The terms “reducing” and “decreasing” are used interchangeably hereinand indicate any change that is less than the original. “Reducing” and“decreasing” are relative terms, requiring a comparison between pre- andpost-measurements. “Reducing” and “decreasing” include completedepletions. The term “modulating” T cell maturation, as used herein,refers to the use of any intervention described herein to control thematuration and/or differentiation of one or more cells such as T cells.For example, modulating refers to inactivating, delaying or inhibiting Tcell maturation. In another example, modulating refers to acceleratingor promoting T cell maturation. The term “delaying or inhibiting T cellmaturation” refers to maintaining one or more T cells in an immature orundifferentiated state. For example, “delaying or inhibiting T cellmaturation” may refer to maintaining T cells in a naïve or T_(CM) state,as opposed to progressing to a TEM or TEFF state. In addition, “delayingor inhibiting T cell maturation” may refer to increasing or enrichingthe overall percentage of immature or undifferentiated T cells (e.g.,naïve T cells and/or T_(CM) cells) within a mixed population of T cells.The state of a T cell (e.g., as mature or immature) may be determined,e.g., by screening for the expression of various genes and the presenceof various proteins expressed on the surface of the T cells. Forexample, the presence of one or more marker selected from the groupconsisting of L-selectin (CD62L+), IL-7R-α, CD132, CR7, CD45RA, CD45RO,CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and any combination thereof maybe indicative of less mature, undifferentiated T cells.

“Treatment” or “treating” of a subject/patient refers to any type ofintervention or process performed on, or the administration of one ormore T cells prepared by the present application to, the subject/patientwith the objective of reversing, alleviating, ameliorating, inhibiting,slowing down or preventing the onset, progression, development, severityor recurrence of a symptom, complication or condition, or biochemicalindicia associated with a disease. In one aspect, “treatment” or“treating” includes a partial remission. In another aspect, “treatment”or “treating” includes a complete remission.

Various aspects of the application are described in further detail inthe following subsections.

Patients with B-cell malignancies bearing high levels of circulatingCD19-expressing tumor cells represent a population with very high unmetneed. For example, Mantle Cell Lymphoma (MCL) is challenging to treat inthe relapsed or refractory setting and remains incurable. Nostandard-of-care exists for second-line and higher chemotherapy.Treatment options include cytotoxic chemotherapy, proteasome inhibitors,immunomodulatory drugs, tyrosine kinase inhibitors, and stem celltransplant (both autologous [ASCT] and allogenic stem cell transplant[allo-SCT]). The choice of regimen is influenced by prior therapy,comorbidities and tumor chemosensitivity. Despite the high initialresponse rates observed with Bruton's tyrosine kinase inhibitor (BTKinhibitors), most patients will eventually develop progressive disease.New therapeutic strategies are needed to improve the dismal prognosis ofpatients with r/r MCL whose disease has not been effectively controlledwith chemo-immunotherapy, stem cell transplant and the BTK inhibitors.

The anti-CD19 CAR T-cell therapy or product used in CD19 CAR-T may bemanufactured from the patient's own T cells, via leukapheresis suitablefor B-cell malignancies with circulating tumor cell burden to minimizethe CD19-expressing tumor cells in the final product. The T cells fromthe harvested leukocytes from the leukapheresis product may be enrichedby selection for CD4+/CD8+ T cells, activated with anti-CD3 andanti-CD28 antibodies, and/or transduced with a viral vector containingan anti-CD19 CAR gene. More details of the method may be found inPCT/US2015/014520 published as WO2015/120096 and in PCT/US2016/057983published as WO2017/070395. In one embodiment, the cells are not treatedwith AKT inhibitors, IL-7, and IL-15. These engineered T cells may bepropagated to generate a sufficient number of cells to achieve atherapeutic effect. Such process removes CD19-expressing malignant andnormal B cells, which may reduce activation, expansion, and exhaustionof the anti-CD19 CAR T cells.

The activation, transduction, and/or expansion of immune cells may beconducted at any suitable time which allows for the production of (i) asufficient number of cells in the population of engineered immune cellsfor at least one dose for administering to a patient, (ii) a populationof engineered immune cells with a favorable proportion of juvenile cellscompared to a typical longer process, or (iii) both (i) and (ii). Thesuitable time may factor several parameters, including the population ofone or more cells, the cell surface receptor expressed by the immunecells, the vector used, the dose that is needed to have a therapeuticeffect, and/or other variables. The time for activation may be 0 day, 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days, 21 days, or more than 21 days. The time foractivation according to the method of present application would bereduced compared to expansion methods known in the art. For example, thetime for activation may be shorter by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, or may be shorter by more than75%. Further, the time for expansion may be 0 day, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days,12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days,20 days, 21 days, or more than 21 days. The time for expansion accordingto the method of present application would be reduced compared toexpansion methods known in the art. For example, the time for expansionmay be shorter by at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, or may be shorter by more than 75%. In one embodiment, thetime for cell expansion is about 3 days, and the time from enrichment ofthe population of cells to producing the engineered immune cells isabout 6 days.

The delay or inhibition of the maturation or differentiation of the oneor more T cells or DC cells may be measured by any methods known in theart. For example, the delay or inhibition of the maturation ordifferentiation of the one or more T cells or DC cells may be measuredby detecting the presence of one or biomarker. The presence of the oneor more biomarker may be detected by any method known in the art,including, but not limited to, immunohistochemistry and/orfluorescence-activated cell sorting (FACS). The one or more biomarker isselected from the group consisting of L-selectin (CD62L⁺), IL-7Rα,CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, orany combination thereof. In certain aspects, the delay or inhibition ofthe maturation or differentiation of the one or more T cells or DC cell)may be measured by detecting the presence of one or more of L-selectin(CD62L⁺), IL-7Rα, and CD132. One of skill in the art would recognizethat though the present methods may increase the relative proportion ofimmature and undifferentiated T cells or DC cells in a population ofcollected cells, some mature and differentiated cells may still bepresent. As a result, the delay or inhibition of the maturation ordifferentiation of the one or more T cells or DC cells may be measuredby calculating the total percent of immature and undifferentiated cellsin a cell population before and after exposing one or more cellsobtained from a donor subject to hypoxic culture conditions with orwithout pressures above atmospheric pressure. The methods disclosedherein may increase the percentage of immature and undifferentiated Tcells in a T cell population.

The methods described herein further comprise stimulating the populationof cells such as lymphocytes with one or more T-cell stimulating agentsto produce a population of activated T cells under a suitable condition.Any combination of one or more suitable T-cell stimulating agents may beused to produce a population of activated T cells including, including,but not limited to, an antibody or functional fragment thereof whichtargets a T-cell stimulatory or co-stimulatory molecule (e.g., anti-CD2antibody, anti-CD3 antibody (such as OKT-3), anti-CD28 antibody, or afunctional fragment thereof), or any other suitable mitogen (e.g.,tetradecanoyl phorbol acetate (TPA), phytohaemagglutinin (PHA),concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen(PWM)), or a natural ligand to a T-cell stimulatory or co-stimulatorymolecule.

The suitable condition for stimulating or activating the population ofimmune cells as described herein further include a temperature, for anamount of time, and/or in the presence of a level of CO₂. Thetemperature for stimulation may be about 34° C., about 35° C., about 36°C., about 37° C., or about 38° C., about 34-38° C., about 35-37° C.,about 36-38° C., about 36-37° C. or about 37° C.

Another condition for stimulating or activating the population of immunecells as described herein may further include a time for stimulation oractivation. The time for stimulation is about 24-72 hours, about 24-36hours, about 30-42 hours, about 36-48 hours, about 40-52 hours, about42-54 hours, about 44-56 hours, about 46-58 hours, about 48-60 hours,about 54-66 hours, or about 60-72 hours, about 44-52 hours, about 40-44hours, about 40-48 hours, about 40-52 hours, or about 40-56 hours. Inone embodiment, the time for stimulation is about 48 hours or at leastabout 48 hours.

Other conditions for stimulating or activating the population of immunecells as described herein may further include a CO₂ Level. The level ofCO₂ for stimulation is about 1.0-10% CO₂, about 1.0%, about 2.0%, about3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about9.0%, or about 10.0% CO₂, about 3-7% CO₂, about 4-6% CO₂, about 4.5-5.5%CO₂. In one embodiment, the level of CO₂ for stimulation is about 5%CO₂.

The conditions for stimulating or activating the population of immunecells may further comprise a temperature, for an amount of time forstimulation, and/or in the presence of a level of CO₂ in anycombination. For example, the step of stimulating the population ofimmune cells may comprise stimulating the population of immune cellswith one or more immune cell stimulating agents at a temperature ofabout 36-38° C., for an amount of time of about 44-52 hours, and in thepresence of a level of CO₂ of about 4.5-5.5% CO₂. The one or more immunecells of the present application may be administered to a subject foruse in immune or cell therapy. Accordingly, the one or more immune cellsmay be collected from a subject in need of a immune or cell therapy.Once collected, the one or more immune cells may be processed for anysuitable period of time before being administered to a subject.

The concentration, amount, or population of lymphocytes or resultingproduct made by the methods herein is about 1.0-10.0×10⁶ cells/mL. Incertain aspects, the concentration is about 1.0-2.0×10⁶ cells/mL, about1.0-3.0×10⁶ cells/mL, about 1.0-4.0×10⁶ cells/mL, about 1.0-5.0×10⁶cells/mL, about 1.0-6.0×10⁶ cells/mL, about 1.0-7.0×10⁶ cells/mL, about1.0-8.0×10⁶ cells/mL, 1.0-9.0×10⁶ cells/mL, about 1.0-10.0×10⁶ cells/mL,about 1.0-1.2×10⁶ cells/mL, about 1.0-1.4×10⁶ cells/mL, about1.0-1.6×10⁶ cells/mL, about 1.0-1.8×10⁶ cells/mL, about 1.0-2.0×10⁶cells/mL, at least about 1.0×10⁶ cells/mL, at least about 1.1×10⁶cells/mL, at least about 1.2×10⁶ cells/mL, at least about 1.3×10⁶cells/mL, at least about 1.4×10⁶ cells/mL, at least about 1.5×10⁶cells/mL, at least about 1.6×10⁶ cells/mL, at least about 1.7×10⁶cells/mL, at least about 1.8×10⁶ cells/mL, at least about 1.9×10⁶cells/mL, at least about 2.0×10⁶ cells/mL, at least about 4.0×10⁶cells/mL, at least about 6.0×10⁶ cells/mL, at least about 8.0×10⁶cells/mL, or at least about 10.0×10⁶ cells/mL.

An anti-CD3 antibody (or functional fragment thereof), an anti-CD28antibody (or functional fragment thereof), or a combination of anti-CD3and anti-CD28 antibodies may be used in accordance with the step ofstimulating the population of lymphocytes, together or independently ofexposing one or more cells obtained from a donor subject to hypoxicculture conditions with or without pressures above atmospheric pressure.Any soluble or immobilized anti-CD2, anti-CD3 and/or anti-CD28 antibodyor functional fragment thereof may be used (e.g., clone OKT3 (anti-CD3),clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293(anti-CD28), clone 15E8 (anti-CD28)). In some aspects, the antibodiesmay be purchased commercially from vendors known in the art including,but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3pure 1 mg/mL, Part No. 170-076-116), and eBioscience, Inc. Further, oneskilled in the art would understand how to produce an anti-CD3 and/oranti-CD28 antibody by standard methods. In some aspect, the one or moreT cell stimulating agents that are used in accordance with the step ofstimulating the population of lymphocytes include an antibody orfunctional fragment thereof which targets a T-cell stimulatory orco-stimulatory molecule in the presence of a T cell cytokine. In oneembodiment, the one or more T cell stimulating agents include ananti-CD3 antibody and IL-2. In certain embodiment, the T cellstimulating agent includes an anti-CD3 antibody at a concentration of 50ng/mL. The concentration of anti-CD3 antibody is about 20 ng/mL-100ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL,about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about100 ng/mL. In an alternative aspect, T cell activation is not needed.

The methods described herein further comprise transducing the populationof activated immune cells with a viral vector comprising a nucleic acidmolecule which encodes the cell surface receptor, using a single cycleor more of viral transduction to produce a population of transducedimmune cells. Several recombinant viruses have been used as viralvectors to deliver genetic material to a cell. Viral vectors that may beused in accordance with the transduction step may be any ecotropic oramphotropic viral vector including, but not limited to, recombinantretroviral vectors, recombinant lentiviral vectors, recombinantadenoviral vectors, and recombinant adeno-associated viral (AAV)vectors. The method further comprises transducing the one or more immunecells with a retrovirus. In one aspect, the viral vector used totransduce the population of activated immune cells is an MSGV1 gammaretroviral vector. In one embodiment, the viral vector used to transducethe population of activated immune cells is the PG13-CD19-H3 Vectordescribed by Kochenderfer, J. Immunother. 32(7): 689-702 (2009).According to one aspect of this aspect, the viral vector is grown in asuspension culture in a medium which is specific for viral vectormanufacturing referred to herein as a viral vector inoculum. Anysuitable growth media and/or supplements for growing viral vectors maybe used in the viral vector inoculum in accordance with the methodsdescribed herein. According to some aspects, the viral vector inoculumis then added to the serum-free culture media described below during thetransduction step. In some aspect, the one or more immune cells may betransduced with a retrovirus. In one embodiment, the retroviruscomprises a heterologous gene encoding a cell surface receptor. Inanother embodiment, the cell surface receptor may bind an antigen on thesurface of a target cell, e.g., on the surface of a tumor cell. Inaddition to optionally exposing one or more cells obtained from a donorsubject to hypoxic culture conditions with or without pressures aboveatmospheric pressure, the conditions for transducing the population ofactivated immune cells as described herein may comprise a specific time,at a specific temperature and/or in the presence of a specific level ofCO₂. The temperature for transduction is about 34° C., about 35° C.,about 36° C., about 37° C., or about 38° C., about 34-38° C., about35-37° C., about 36-38° C., about 36-37° C. In one embodiment, thetemperature for transduction is about 37° C. The predeterminedtemperature for transduction may be about 34° C., about 35° C., about36° C., about 37° C., about 38° C., or about 39° C., about 34-39° C.,about 35-37° C. In one embodiment, the predetermined temperature fortransduction may be from about 36-38° C., about 36-37° C. or about 37°C. The time for transduction is about 12-36 hours, about 12-16 hours,about 12-20 hours, about 12-24 hours, about 12-28 hours, about 12-32hours, about 20 hours or at least about 20 hours, is about 16-24 hours,about 14 hours, at least about 16 hours, at least about 18 hours, atleast about 20 hours, at least about 22 hours, at least about 24 hours,or at least about 26 hours. The level of CO₂ for transduction is about1.0-10% CO₂, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%,about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0% CO₂, about3-7% CO₂, about 4-6% CO₂, about 4.5-5.5% CO₂, or about 5% CO₂.

Transducing the population of activated immune cells as described hereinmay be performed for a period of time, at certain temperature and/or inthe presence of a specific level of CO₂ in any combination: atemperature of about 36-38° C., for an amount of time of about 16-24hours, and in the presence of a level of CO₂ of about 4.5-5.5% CO₂ Theimmune cells may be prepared by the combination of any one of themethods of the application with any manufacturing method of preparing Tcells for immunotherapy, including, without limitation, those describedin PCT Publications Nos. WO2015/120096 and WO2017/070395, which areherein incorporated by reference in their totality for the purposes ofdescribing these methods; any and all methods used in the preparation ofAxicabtagene ciloleucel or Yescarta®; any and all methods used in thepreparation of Tisagenlecleucel/Kymriah™; any and all methods used inthe preparation of “off-the-shelf” T cells for immunotherapy; and anyother methods of preparing lymphocytes for administration to humans. Themanufacturing process may be adapted to remove circulating tumor cellsfrom the cells obtained from the patient.

CAR-T cells may be engineered to express other molecules and may be ofany one of the following exemplary types or others available in the art:first, second, third, fourth, fifth, or more CAR-T cells; Armored CAR-Tcells, Motile CAR-T cells, TRUCK T-cells, Switch receptor CAR-T cells;Gene edited CAR T-cells; dual receptor CAR T-cells; suicide CAR T-cells,drug-inducible CAR-T cells, synNotch inducible CAR T-cells; andinhibitory CAR T-cells. In one aspect, the T cells are autologousT-cells. In one aspect, the T cells are autologous stem cells (forautologous stem cell therapy or ASCT). In one aspect, the T cells arenon-autologous T-cells.

The cells (such as immune cells or T cells) are genetically modifiedfollowing isolation or selection using known methods or activated and/orexpanded (or differentiated in the case of progenitors) in vitro priorto being genetically modified. The immune cells, e.g., T cells, aregenetically modified with the chimeric antigen receptors describedherein (e.g., transduced with a viral vector comprising one or morenucleotide sequences encoding a CAR) and activated and/or expanded invitro. Methods for activating and expanding T cells may be found in U.S.Pat. Nos. 6,905,874; 6,867,041; and 6,797,514; and PCT Publication No.WO 2012/079000, which are hereby incorporated by reference in theirentirety. Generally, such methods may include contacting PBMC orisolated T cells with a stimulatory agent and costimulatory agent, suchas anti-CD3 and/or anti-CD28 antibodies, that may be attached to a beador other surface, in a culture medium with certain cytokines, such asIL-2. The Dynabeads® system, a CD3/CD28 activator/stimulator system forphysiological activation of human T cells may be used. The T cells maybe activated and stimulated to proliferate with suitable feeder cells,antibodies and/or cytokines as described in U.S. Pat. Nos. 6,040,177 and5,827,642 and PCT Publication No. WO 2012/129514, which are herebyincorporated by reference in their entirety.

The cell surface receptor that is expressed by the engineered immunecells may be any antigen or molecule to be targeted by CAR, such as ananti-CD19 CAR, FMC63-28Z CAR, or FMC63-CD828BBZ CAR (Kochenderfer etal., J Immunother. 2009, 32(7): 689; Locke et al., Blood 2010,116(20):4099, the subject matter of both of which is hereby incorporatedby reference. In certain aspects, the predetermined dose of engineeredimmune cells may be more than about 1 million to less than about 3million transduced engineered T cells/kg. In one embodiment, thepredetermined dose of engineered T cells may be more than about 1million to about 2 million transduced engineered T cells per kilogram ofbody weight (cells/kg). The predetermined dose of engineered T cells maybe more than 1 million to about 2 million, at least about 2 million toless than about 3 million transduced engineered T cells per kilogram ofbody weight (cells/kg). In one embodiment, the predetermined dose ofengineered T cells may be about 2 million transduced engineered Tcells/kg. In another embodiment, the predetermined dose of engineered Tcells may be at least about 2 million transduced engineered T cells/kg.Examples of the predetermined dose of engineered T cells may be about2.0 million, about 2.1 million, about 2.2 million, about 2.3 million,about 2.4 million, about 2.5 million, about 2.6 million, about 2.7million, about 2.8 million, or about 2.9 million transduced engineered Tcells/kg.

The methods described herein comprise increasing or enriching thepopulation of transduced one or more immune cells for a period of timeto produce a population of engineered immune cells. The time forexpansion may be any suitable time which allows for production of (i) asufficient number of cells in the population of engineered immune cellsfor at least one dose for administering to a patient, (ii) a populationof engineered immune cells with a favorable proportion of juvenile cellscompared to a typical longer process, or (iii) both (i) and (ii). Thistime will depend on the cell surface receptor expressed by the immunecells, the vector used, the dose that is needed to have a therapeuticeffect, and other variables. The predetermined time for expansion may be0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In oneembodiment, the time for expansion of the present method is reducedcompared to those known in the art. For example, the predetermined timefor expansion may be shorter by at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, or may be shorter by more than 75%. In oneexample, the time for expansion is about 3 days, and the time fromenrichment of the population of lymphocytes to producing the engineeredimmune cells is about 6 days.

The conditions for expanding the population of transduced immune cellsmay include a temperature and/or in the presence of a level of CO₂. Incertain aspects, the temperature is about 34° C., about 35° C., about36° C., about 37° C., or about 38° C., about 35-37° C., about 36-37° C.,or about 37° C. The level of CO₂ is 1.0-10% CO₂, about 1.0%, about 2.0%,about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%,about 9.0%, about 10.0% CO₂, about 4.5-5.5% CO₂, about 5% CO₂, about3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO₂.

Each step of the methods described herein may be performed in a closedsystem. The closed system may be a closed bag culture system, using anysuitable cell culture bags (e.g., Miltenyi Biotec MACS® GMP CellDifferentiation Bags, Origen Biomedical PermaLife Cell Culture bags).The cell culture bags used in the closed bag culture system may becoated with a recombinant human fibronectin fragment during thetransduction step. The recombinant human fibronectin fragment mayinclude three functional domains: a central cell-binding domain,heparin-binding domain II, and a CS1-sequence. The recombinant humanfibronectin fragment may be used to increase gene efficiency ofretroviral transduction of immune cells by aiding co-localization oftarget cells and viral vector. In one embodiment, the recombinant humanfibronectin fragment is RETRONECTIN® (Takara Bio, Japan). The cellculture bags are coated with recombinant human fibronectin fragment at aconcentration of about 1-60 μg/mL or about 1-40 μg/mL, about 1-20 μg/mL,20-40 μg/mL, 40-60 μg/mL, about 1 μg/mL, about 2 μg/mL, about 3 μg/mL,about 4 μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8μg/mL, about 9 μg/mL, about 10 μg/mL, about 11 μg/mL, about 12 μg/mL,about 13 μg/mL, about 14 μg/mL, about 15 μg/mL, about 16 μg/mL, about 17μg/mL, about 18 μg/mL, about 19 μg/mL, about 20 μg/mL, about 2-5 μg/mL,about 2-10 μg/mL, about 2-20 μg/mL, about 2-25 μg/mL, about 2-30 μg/mL,about 2-35 μg/mL, about 2-40 μg/mL, about 2-50 μg/mL, about 2-60 μg/mL,at least about 2 μg/mL, at least about 5 μg/mL, at least about 10 μg/mL,at least about 15 μg/mL, at least about 20 μg/mL, at least about 25μg/mL, at least about 30 μg/mL, at least about 40 μg/mL, at least about50 μg/mL, or at least about 60 μg/mL recombinant human fibronectinfragment. In one embodiment, the cell culture bags are coated with atleast about 10 μg/mL recombinant human fibronectin fragment. The cellculture bags used in the closed bag culture system may optionally beblocked with human albumin serum (HSA) during the transduction step. Inanother embodiment, the cell culture bags are not blocked with HSAduring the transduction step.

The population of engineered immune cells produced by the methodsdescribed above may optionally be cryopreserved so that the cells may beused later. A method for cryopreservation of a population of engineeredimmune cells also is provided herein. Such a method may include a stepof washing and concentrating the population of engineered immune cellswith a diluent solution. For example, the diluent solution is normalsaline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose/0.45% NaCl salinesolution (D5), human serum albumin (HSA), or a combination thereof.Also, HSA may be added to the washed and concentrated cells for improvedcell viability and cell recovery after thawing. In another aspect, thewashing solution is normal saline and washed and concentrated cells aresupplemented with HSA (5%). The method may also include a step ofgenerating a cryopreservation mixture, wherein the cryopreservationmixture includes the diluted population of cells in the diluent solutionand a suitable cryopreservative solution. The cryopreservative solutionmay be any suitable cryopreservative solution including, but not limitedto, CryoStor10 (BioLife Solution), mixed with the diluent solution ofengineered immune cells at a ratio of 1:1 or 2:1. HSA may be added toprovide a final concentration of about 1.0-10%, about 1.0%, about 2.0%,about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%,about 9.0%, about 10.0%, about 1-3% HSA, about 1-4% HSA, about 1-5% HSA,about 1-7% HSA, about 2-4% HSA, about 2-5% HSA, about 2-6% HSA, about2-7% HAS or about 2.5% HSA in the cryopreserved mixture.Cryopreservation of a population of engineered immune cells may comprisewashing cells with 0.9% normal saline, adding HSA at a finalconcentration of 5% to the washed cells, and diluting the cells 1:1 withCryoStor™ CS10 (for a final concentration of 2.5% HSA in the finalcryopreservation mixture). In some aspect, the method also includes astep of freezing the cryopreservation mixture. Also, thecryopreservation mixture is frozen in a controlled rate freezer using adefined freeze cycle at a cell concentration of between about 1×10⁶ toabout 1.5×10⁷ cells/mL of cryopreservation mixture. The method may alsoinclude a step of storing the cryopreservation mixture in vapor phaseliquid nitrogen.

The population of engineered immune cells produced by the methodsdescribed herein may be cryopreserved at a predetermined dose. Thepredetermined dose may be a therapeutically effective dose, which may beany therapeutically effective dose as provided below. The predetermineddose of engineered immune cells may depend on the cell surface receptorthat is expressed by the immune cells (e.g., the affinity and density ofthe cell surface receptors expressed on the cell), the type of targetcell, the nature of the disease or pathological condition being treated,or a combination of both.

In one embodiment, the population of engineered T cells may becryopreserved at a predetermined dose of about 1 million engineered Tcells per kilogram of body weight (cells/kg). In certain embodiment, thepopulation of engineered T cells may be cryopreserved at a predetermineddose of from about 500,000 to about 1 million engineered T cells/kg. Incertain embodiment, the population of engineered T cells may becryopreserved at a predetermined dose of at least about 1 million, atleast about 2 million, at least about 3 million, at least about 4million, at least about 5 million, at least about 6 million, at leastabout 7 million, at least about 8 million, at least about 9 million, atleast about 10 million engineered T cells/kg. In other aspects, thepopulation of engineered T cells may be cryopreserved at a predetermineddose of less than 1 million cells/kg, 1 million cells/kg, 2 millioncells/kg, 3 million cells/kg, 4 million cells/kg, 5 million cells/kg, 6million cells/kg, 7 million cells/kg, 8 million cells/kg, 9 millioncells/kg, 10 million cells/kg, more than 10 million cells/kg, more than20 million cells/kg, more than 30 million cells/kg, more than 40 millioncells/kg, more than 50 million cells/kg, more than 60 million cells/kg,more than 70 million cells/kg, more than 80 million cells/kg, more than90 million cells/kg, or more than 100 million cells/kg. In certainaspects, the population of engineered T cells may be cryopreserved at apredetermined dose of from about 1 million to about 2 million engineeredT cells/kg. The population of engineered T cells may be cryopreserved ata predetermined dose between about 1 million cells to about 2 millioncells/kg, about 1 million cells to about 3 million cells/kg, about 1million cells to about 4 million cells/kg, about 1 million cells toabout 5 million cells/kg, about 1 million cells to about 6 millioncells/kg, about 1 million cells to about 7 million cells/kg, about 1million cells to about 8 million cells/kg, about 1 million cells toabout 9 million cells/kg, about 1 million cells to about 10 millioncells/kg. The predetermined dose of the population of engineered T cellsmay be calculated based on a subject's body weight. In one example, thepopulation of engineered T cells may be cryopreserved in about 0.5-200mL of cryopreservation media. Additionally, the population of engineeredT cells may be cryopreserved in about 0.5 mL, about 1.0 mL, about 5.0mL, about 10.0 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL,about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL,about 10-30 mL, about 10-50 mL, about 10-70 mL, about 10-90 mL, about50-70 mL, about 50-90 mL, about 50-110 mL, about 50-150 mL, or about100-200 mL of cryopreservation media. In certain aspects, the populationof engineered T cells may be preferably cryopreserved in about 50-70 mLof cryopreservation media.

In one embodiment, at least one of (a) contacting the population ofimmune cells with exogenous IL-2, exogenous IL-7, exogenous IL-15,and/or other cytokine(s), (b) stimulating the population of immune cells(c) transducing the population of activated immune cells, and (d)expanding the population of transduced immune cells is performed using aserum-free culture medium which is free from added serum. In someaspect, each of (a) to (d) is performed using a serum-free culturemedium which is free from added serum. As referred to herein, the term“serum-free media” or “serum-free culture medium” means that the growthmedia used is not supplemented with serum (e.g., human serum or bovineserum). In other words, no serum is added to the culture medium as anindividually separate and distinct ingredient for the purpose ofsupporting the viability, activation and grown of the cultured cells.Any suitable immune cell growth media may be used for culturing thecells in suspension in accordance with the methods described herein. Forexample, an immune cell growth media may include, but is not limited to,a sterile, low glucose solution that includes a suitable amount ofbuffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. Inone aspect, the T cell growth media is OPTMIZER™ (Life Technologies). Incontrast to typical methods for producing engineered immune cells, themethods described herein may use culture medium that is not supplementedwith serum (e.g., human or bovine).

The application provides various methods of treatment of cancer with Tcells. In one aspect, the T cells are CAR-T cells against CD19, whichmay be prepared by the combination of any one of the methods of theapplication with any step of the manufacturing method of preparing Tcells for immunotherapy, including, without limitation, those describedin PCT Publication Nos. WO2015/120096 and WO2017/070395, both of whichare herein incorporated by reference in their totality for the purposesof describing these methods; any and all methods used in the preparationof Axicabtagene ciloleucel or Yescarta®; any and all methods used in thepreparation of Tisagenlecleucel/Kymriah™; any and all methods used inthe preparation of “off-the-shelf” T cells for immunotherapy; and anyother methods of preparing lymphocytes for administration to humans. Insome aspect, the manufacturing process is adapted to specifically removecirculating tumor cells from the cells obtained from the patient.

In one aspect, the T cells are the CD19 CAR-T cells, prepared by themethod described in PCT/US2016/057983. In one embodiment, a populationof T cells that is depleted of circulating tumor cells is prepared fromleukapheresis products. These cells may be prepared as described inPCT/US2016/057983 and are further described herein as CD19 CAR-T cells.Briefly, CD19 CAR-T is an autologous CAR T-cell product in which asubject's T cells are engineered to express receptors consisting of asingle-chain antibody fragment against CD19 linked to CD28 and CD3ζactivating domains that result in elimination of CD19-expressing cells.Following CAR engagement with CD19⁺ target cells, the CD3ζ domainactivates the downstream signaling cascade that leads to T-cellactivation, proliferation, and acquisition of effector functions, suchas cytotoxicity. The intracellular signaling domain of CD28 provides acostimulatory signal that function with the primary CD3ζ signal toaugment T-cell function, including interleukin (IL)-2 production.Together, these signals may stimulate proliferation of the CAR T cellsand direct killing of target cells. In addition, activated T cells maysecrete cytokines, chemokines, and other molecules that may recruit andactivate additional antitumor immune cells. The anti-CD19 CAR in theCD19 CAR-T cells may comprise FMC63-28Z.

Due to the presence of circulating tumor cells in certain cancers, themanufacture of CD19 CAR-T includes a CD4⁺ and CD8⁺ T-cell enrichmentstep. The T-cell enrichment or isolation step may reduce circulatingCD19-expressing tumor cells in leukapheresis material, and may relate tothe activation, expansion, and exhaustion of the anti-CD19 CAR T cellsduring manufacturing.

The methods described herein may enhance the treatment outcome oreffectiveness of a immune or cell therapy), which may be an adoptive Tcell therapy selected from the group consisting of tumor-infiltratinglymphocyte (TIL) immunotherapy, autologous cell therapy, engineeredautologous cell therapy (eACT™), allogeneic T cell transplantation,non-T cell transplantation, and any combination thereof. Adoptive T celltherapy broadly includes any method of selecting, enriching in vitro,and administering to a patient autologous or allogeneic T cells thatrecognize and are capable of binding tumor cells. TIL immunotherapy is atype of adoptive T cell therapy, wherein lymphocytes capable ofinfiltrating tumor tissue are isolated, enriched in vitro, andadministered to a patient. The TIL cells may be either autologous orallogeneic. Autologous cell therapy is an adoptive T cell therapy thatinvolves isolating T cells capable of targeting tumor cells from apatient, enriching the T cells in vitro, and administering the T cellsback to the same patient. Allogeneic T cell transplantation may includetransplant of naturally occurring T cells expanded ex vivo orgenetically engineered T cells. Engineered autologous cell therapy, asdescribed in more detail above, is an adoptive T cell therapy wherein apatient's own lymphocytes are isolated, genetically modified to expressa tumor targeting molecule, expanded in vitro, and administered back tothe patient. Non-T cell transplantation may include autologous orallogeneic therapies with non-T cells such as, but not limited to,natural killer (NK) cells.

The immune cell therapy of the present application is engineeredAutologous Cell Therapy (eACT™). According to this aspect, the methodmay include collecting immune cells from a donor. The isolated immunecells may then be contacted with an exogenous activation reagent (e.g.,cytokine), expanded, and engineered to express a chimeric antigenreceptor (“engineered CAR T cells”) or T cell receptor (“engineered TCRT cells”). In some aspect, the engineered immune cells treat a tumor inthe subject. For example, the one or more immune cells are transducedwith a retrovirus comprising a heterologous gene encoding a cell surfacereceptor. In one embodiment x, the cell surface receptor is capable ofbinding an antigen on the surface of a target cell, e.g., on the surfaceof a tumor cell. In some embodiment, the cell surface receptor is achimeric antigen receptor or a T cell receptor. In another embodiment,the one or more immune cells may be engineered to express a chimericantigen receptor. The chimeric antigen receptor may comprise a bindingmolecule to a tumor antigen. The binding molecule may be an antibody oran antigen binding molecule thereof. For example, the antigen bindingmolecule may be selected from scFv, Fab, Fab′, Fv, F(ab′)2, and dAb, andany fragments or combinations thereof. The chimeric antigen receptor mayfurther comprise a hinge region. The hinge region may be derived fromthe hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, orCD8 alpha. In one embodiment, the hinge region is derived from the hingeregion of IgG4. The chimeric antigen receptor may also comprise atransmembrane domain. The transmembrane domain may be a transmembranedomain of any transmembrane molecule that is a co-receptor on immunecells or a transmembrane domain of a member of the immunoglobulinsuperfamily. In certain embodiment, the transmembrane domain is derivedfrom a transmembrane domain of CD28, CD28T, CD8 alpha, CD4, or CD19. Inanother embodiment, the transmembrane domain comprises a domain derivedfrom a CD28 transmembrane domain. In another embodiment, thetransmembrane domain comprises a domain derived from a CD28Ttransmembrane domain. The chimeric antigen receptor may further compriseone or more costimulatory signaling regions. For example, thecostimulatory signaling region may be a signaling region of CD28, CD28T,OX-40, 41BB, CD27, inducible T cell costimulator (ICOS), CD3 gamma, CD3delta, CD3 epsilon, CD247, Ig alpha (CD79a), or Fc gamma receptor. Infurther embodiment, the costimulatory signaling region is a CD28signaling region. In another embodiment, the costimulatory signalingregion is a CD28T signaling region. In additional embodiment, thechimeric antigen receptor further comprises a CD3 zeta signaling domain.

In some aspects, the tumor antigen is selected from 707-AP (707 alanineproline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigenrecognized by T4 cells), BAGE (B antigen; b-catenin/m,b-catenin/mutated), BCMA (B cell maturation antigen), Bcr-abl(breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19(cluster of differentiation 19), CD20 (cluster of differentiation 20),CD22 (cluster of differentiation 22), CD30 (cluster of differentiation30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster ofdifferentiation 44, exons 7/8), CAMEL (CTL-recognized antigen onmelanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8(caspase-8), CDC27m (cell-division cycle 27 mutated), CDK4/m(cycline-dependent kinase 4 mutated), CEA (carcinoembryonic antigen), CT(cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiationantigen melanoma), EGFR (epidermal growth factor receptor), EGFRvIII(epidermal growth factor receptor, variant III), EGP-2 (epithelialglycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4(erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M(elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6/acutemyeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (Fetalacetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2(disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V(N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100 kD), HAGE(helicose antigen), HER-2/neu (human epidermal receptor-2/neurological;also known as EGFR2), HLA-A (human leukocyte antigen-A) HPV (humanpapilloma virus), HSP70-2M (heat shock protein 70-2 mutated), HST-2(human signet ring tumor-2), hTERT or hTRT (human telomerase reversetranscriptase), iCE (intestinal carboxyl esterase), IL-13R-a2(Interleukin-13 receptor subunit alpha-2), KIAA0205, KDR (kinase insertdomain receptor), κ-light chain, LAGE (L antigen), LDLR/FUT (low densitylipid receptor/GDP-L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase),LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE(melanoma antigen), MAGE-A1 (Melanoma-associated antigen 1), MAGE-A3,MAGE-A6, mesothelin, Murine CMV infected cells, MART-1/Melan-A (melanomaantigen recognized by T cells-1/Melanoma antigen A), MC1R (melanocortin1 receptor), Myosin/m (myosin mutated), MUC1 (mucin 1), MUM-1, -2, -3(melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patientM88), NKG2D (Natural killer group 2, member D) ligands, NY-BR-1 (NewYork breast differentiation antigen 1), NY-ESO-1 (New York esophagealsquamous cell carcinoma-1), oncofetal antigen (h5T4), P15 (protein 15),p190 minor bcr-abl (protein of 190 KD bcr-abl), Pml/RARa (promyelocyticleukaemia/retinoic acid receptor a), PRAME (preferentially expressedantigen of melanoma), PSA (prostate-specific antigen), PSCA (Prostatestem cell antigen), PSMA (prostate-specific membrane antigen), RAGE(renal antigen), RU1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcomaantigen), SART-1 or SART-3 (squamous antigen rejecting tumor 1 or 3),SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor-associatedantigen), TAG-72 (Tumor-associated glycoprotein 72), TEL/AML1(translocation Ets-family leukemia/acute myeloid leukemia 1), TPI/m(triosephosphate isomerase mutated), TRP-1 (tyrosinase related protein1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2/INT2(TRP-2/intron 2), VEGF-R2 (vascular endothelial growth factor receptor2), WT1 (Wilms' tumor gene), and any combination thereof. In oneembodiment, the tumor antigen is CD19.

The T cell therapy comprises administering to the patient engineered Tcells expressing T cell receptor (“engineered TCR T cells”). The T cellreceptor (TCR) may comprise a binding molecule to a tumor antigen. Insome aspects, the tumor antigen is selected from the group consisting of707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAM CD19, CD20, CD22, CD30,CD33, CD44v7/8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B,DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP,fAchR, G250, GAGE, GD2, GD3, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A, HPV,HSP70-2M, HST-2, hTERT or hTRT, iCE, IL-13R-a2, KIAA0205, KDR, κ-lightchain, LAGE, LDLR/FUT, LeY, L1CAM, MAGE, MAGE-AL mesothelin, Murine CMVinfected cells, MART-1/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3,NA88-A, NKG2D ligands, NY-BR-1, NY-ESO-1, oncofetal antigen, P15, p190minor bcr-abl, Pml/RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE,SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL/AML1, TPI/m, TRP-1,TRP-2, TRP-2/INT2, VEGF-R2, WT1, and any combination thereof.

“CD19-directed genetically modified autologous T cell immunotherapy”refers to a suspension of chimeric antigen receptor (CAR)-positiveimmune cells. An example of such immunotherapy is Clear CAR-T therapy,which uses CAR-T cells that are free of circulating tumor cells andenriched in CD4+/CD8+ T cells. Another example is axicabtageneciloleucel (also known as) YESCARTA™, See Kochenderfer, et al., (JImmunother 2009; 32:689 702). Other non-limiting examples includeJCAR017, JCAR015, JCAR014, Kymriah (tisagenlecleucel), Uppsala U.anti-CD19 CAR (NCT02132624), and UCART19 (Celectis). See Sadelain et al.Nature Rev. Cancer Vol. 3 (2003), Ruella et al., Curr Hematol MaligRep., Springer, N.Y. (2016) and Sadelain et al. Cancer Discovery (April2013) To prepare CD19-directed genetically modified autologous T cellimmunotherapy, a patient's own T cells may be harvested and geneticallymodified ex vivo by retroviral transduction to express a chimericantigen receptor (CAR) comprising a murine anti-CD19 single chainvariable fragment (scFv) linked to CD28 and CD3-zeta co-stimulatorydomains. In some embodiments, the CAR comprises a murine anti-CD19single chain variable fragment (scFv) linked to 4-1BB and CD3-zetaco-stimulatory domain. The anti-CD19 CAR T cells may be expanded andinfused back into the patient, where they may recognize and eliminateCD19-expressing target cells.

In one aspect, the TCR comprises a binding molecule to a viral oncogene.In one embodiment, the viral oncogene is selected from human papillomavirus (HPV), Epstein-Ban virus (EBV), and human T-lymphotropic virus(HTLV). In other embodiment, the TCR comprises a binding molecule to atesticular, placental, or fetal tumor antigen. In one embodiment, thetesticular, placental, or fetal tumor antigen is selected from the groupconsisting of NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanomaantigen (MAGE), and any combination thereof. In another embodiment, theTCR comprises a binding molecule to a lineage specific antigen. Inadditional embodiment, the lineage specific antigen is selected from thegroup consisting of melanoma antigen recognized by T cells 1 (MART-1),gp100, prostate specific antigen (PSA), prostate specific membraneantigen (PSMA), prostate stem cell antigen (PSCA), and any combinationthereof. In certain embodiment, the T cell therapy comprisesadministering to the patient engineered CAR T cells expressing achimeric antigen receptor that binds to CD19 and further comprises aCD28 costimulatory domain and a CD3-zeta signaling region. In additionalembodiment, the T cell therapy comprises administering to a patientKTE-C19. In one aspect, the antigenic moieties also include, but are notlimited to, an Epstein-Barr virus (EBV) antigen (e.g., EBNA-1, EBNA-2,EBNA-3, LMP-1, LMP-2), a hepatitis A virus antigen (e.g., VP1, VP2,VP3), a hepatitis B virus antigen (e.g., HBsAg, HBcAg, HBeAg), ahepatitis C viral antigen (e.g., envelope glycoproteins E1 and E2), aherpes simplex virus type 1, type 2, or type 8 (HSV1, HSV2, or HSV8)viral antigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK,gL. gM, UL20, UL32, US43, UL45, UL49A), a cytomegalovirus (CMV) viralantigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL. gMor other envelope proteins), a human immunodeficiency virus (HIV) viralantigen (glycoproteins gp120, gp41, or p24), an influenza viral antigen(e.g., hemagglutinin (HA) or neuraminidase (NA)), a measles or mumpsviral antigen, a human papillomavirus (HPV) viral antigen (e.g., L1,L2), a parainfluenza virus viral antigen, a rubella virus viral antigen,a respiratory syncytial virus (RSV) viral antigen, or avaricella-zostser virus viral antigen. In such aspects, the cell surfacereceptor may be any TCR, or any CAR which recognizes any of theaforementioned viral antigens on a target virally infected cell. Inother aspects, the antigenic moiety is associated with cells having animmune or inflammatory dysfunction. Such antigenic moieties may include,but are not limited to, myelin basic protein (MBP) myelin proteolipidprotein (PLP), myelin oligodendrocyte glycoprotein (MOG),carcinoembryonic antigen (CEA), pro-insulin, glutamine decarboxylase(GAD65, GAD67), heat shock proteins (HSPs), or any other tissue specificantigen that is involved in or associated with a pathogenic autoimmuneprocess.

The methods disclosed herein may involve a T cell therapy comprising thetransfer of one or more T cells to a patient. The T cells may beadministered at a therapeutically effective amount. For example, atherapeutically effective amount of T cells, e.g., engineered CAR+ Tcells or engineered TCR+ T cells, may be at least about 10⁴ cells, atleast about 10⁵ cells, at least about 10⁶ cells, at least about 10⁷cells, at least about 10⁸ cells, at least about 10⁹, or at least about10¹⁰. In another aspect, the therapeutically effective amount of the Tcells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, isabout 10⁴ cells, about 10⁵ cells, about 10⁶ cells, about 10⁷ cells, orabout 10⁸ cells. In one embodiment, the therapeutically effective amountof the T cells, e.g., engineered CAR+ T cells or engineered TCR+ Tcells, is about 2×10⁶ cells/kg, about 3×10⁶ cells/kg, about 4×10⁶cells/kg, about 5×10⁶ cells/kg, about 6×10⁶ cells/kg, about 7×10⁶cells/kg, about 8×10⁶ cells/kg, about 9×10⁶ cells/kg, about 1×10⁷cells/kg, about 2×10⁷ cells/kg, about 3×10⁷ cells/kg, about 4×10⁷cells/kg, about 5×10⁷ cells/kg, about 6×10⁷ cells/kg, about 7×10⁷cells/kg, about 8×10⁷ cells/kg, or about 9×10⁷ cells/kg. In oneembodiment, the amount of CD19 CAR-T cells is 2×10⁶ cells/kg, with amaximum dose of 2×10⁸ cells for subjects ≥100 kg. In another embodiment,the amount of CD19 CAR-T cells is 0.5×10⁶ cells/kg, with a maximum doseof 0.5×10⁸ cells for subjects ≥100 kg.

The patients may be preconditioned or lymphodepleted prior toadministration of the T cell therapy. The patient may be preconditionedaccording to any methods known in the art, including, but not limitedto, treatment with one or more chemotherapy drug and/or radiotherapy. Insome aspects, the preconditioning may include any treatment that reducesthe number of endogenous lymphocytes, removes a cytokine sink, increasesa serum level of one or more homeostatic cytokines or pro-inflammatoryfactors, enhances an effector function of T cells administered after theconditioning, enhances antigen presenting cell activation and/oravailability, or any combination thereof prior to a T cell therapy. Thepreconditioning may comprise increasing a serum level of one or morecytokines in the subject. The methods further comprise administering achemotherapeutic. The chemotherapeutic may be a lymphodepleting(preconditioning) chemotherapeutic. Beneficial preconditioning treatmentregimens, along with correlative beneficial biomarkers are described inU.S. Pat. No. 9,855,298, which is hereby incorporated by reference inits entirety herein. These describe, e.g., methods of conditioning apatient in need of a T cell therapy comprising administering to thepatient specified beneficial doses of cyclophosphamide (between 200mg/m²/day and 2000 mg/m²/day) and specified doses of fludarabine(between 20 mg/m²/day and 900 mg/m²/day). One such dose regimen involvestreating a patient comprising administering daily to the patient about500 mg/m²/day of cyclophosphamide and about 60 mg/m²/day of fludarabinefor three days prior to administration of a therapeutically effectiveamount of engineered T cells to the patient. In one aspect, theconditioning regimen comprises cyclophosphamide 500 mg/m²+fludarabine 30mg/m² for 3 days. They may be administered at days −4, −3, and −2 or atdays −5, −4, and −3 (day 0 being the day of administration of thecells). In one embodiment, the conditioning regimen comprisescyclophosphamide 200 mg/m², 250 mg/m², 300 mg/m², 400 v, 500 mg/m² dailyfor 2, 3, or 4 days and fludarabine 20 mg/m², 25 mg/m², or 30 mg/m² for2, 3, or 4 days. In one embodiment, and after leukapheresis,conditioning chemotherapy (fludarabine 30 mg/m²/day and cyclophosphamide500 mg/m²/day) is administered on days −5, −4, and −3 prior to anintravenous infusion of a suspension of CD19 CAR-T cells. In someembodiments, the intravenous infusion time is between 15 and 120minutes. In one embodiment, the intravenous infusion time is between 1and 240 minutes. In some embodiments, the intravenous infusion time isup to 30 minutes. In some embodiments, the intravenous infusion time isup to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or up to 100 minutes. In some embodiments, the infusion volumeis between 50 and 100 mL. In some embodiments, the infusion volume isbetween 20 and 100 ml. In some embodiments, the infusion volume is about30, 35, 40, 45, 50, 55, 60, or about 65 ml. In some embodiments, theinfusion volume is about 68 mL. In some embodiments, the suspension hasbeen frozen and is used within 6, 5, 4, 3, 2, 1 hour of thawing. In someembodiments, the suspension has not been frozen. In some embodiments,the immunotherapy is infused from an infusion bag. In some embodiments,the infusion bag is agitated during the infusion. In some embodiments,the immunotherapy is administered within 3 hours after thawing. In someembodiments, the suspension further comprises albumin. In someembodiments, albumin is present in an amount of about 2-3% (v/v). Insome embodiments, albumin is present in an amount of about 2.5% (v/v).In some embodiments, the albumin is present in an amount of about 1%,2%, 3%, 4%, or 5% (v/v). In some embodiments, albumin is human albumin.In some embodiments, the suspension further comprises DMSO. In someembodiments, DMSO is present in an amount of about 4-6% (v/v). In someembodiments, DMSO is present in an amount of about 5% (v/v). In someembodiments, the DMSO is present in an amount of 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, or 10% (v/v).

The methods disclosed herein may be used to treat a cancer in a subject,reduce the size of a tumor, kill tumor cells, prevent tumor cellproliferation, prevent growth of a tumor, eliminate a tumor from apatient, prevent relapse of a tumor, prevent tumor metastasis, induceremission in a patient, or any combination thereof. In certain aspects,the methods may induce a complete response. In other aspects, themethods may induce a partial response.

Cancers that may be treated include tumors that are not vascularized,not yet substantially vascularized, or vascularized. The cancer may alsoinclude solid or non-solid tumors.

In one embodiment, the method may be used to treat a B-cell malignancybearing high levels of circulating CD19-expressing tumor cells and willbe indicated for a distinct patient population with high unmet need.

Exemplary Treatment of MCL

In some embodiments, the malignancy may be mantle cell lymphoma (MCL).MCL is an aggressive subtype of non-Hodgkin lymphoma (NHL). MCL accountsfor approximately 6% of all new cases of NHL in the United States (US)and 5% to 7% of malignant lymphoma in Western Europe The estimatedannual incidence of MCL is approximately 1 to 2 per 100,000 persons inthe US and Europe. MCL is more likely to affect men than women, and themedian age at diagnosis is 68 years. In some embodiments, the r/r MCL isr/r to treatment with allogeneic stem cell transplant (allo-SCT), whichitself can result in durable remission for approximately 25% of patientswith relapsed or refractory (r/r) MCL if their disease was shown to bechemosensitive prior to transplant, but allo-SCT is also associated withtreatment-related mortality rates of up to 40%.

In some embodiments, the r/r MCL is r/r to treatment with bortezomib,lenalidomide, and temsirolimus, which itself results in ORRs rangingfrom 22% to 32%. Bruton's tyrosine kinase (BTK) inhibitors such asibrutinib and acalabrutinib result in ORRs of 68% and 81%, respectively,in patients with r/r MCL. However, most patients progress following BTKinhibitor treatment and have poor outcomes in response to salvagetherapies, with ORRs ranging from 20% to 42%, median durations ofresponse (DORs) ranging from 3 to 5.4 months, and median OS ranging from2.5 to 9 months. In some embodiments, the disclosure provides that CAR Tcell intervention can be used to treat cancers with poor prognosticfactors such as high Ki67 tumor proliferation index expression (≥30% or≥50%) and mutated TP53. In some embodiments, the cancer is MCL. In someembodiments, the MCL morphology is classical, pleomorphic, or blastoid.In some embodiments, the Ki-67 index may be between 5% and 80%. In someembodiments, the Ki-67 index is about 38%. In some embodiments,high-risk patients have a Ki-67 ≥50% and/or TP53 mutation by nextgenerations sequencing. In some embodiments, the patient is aged ≥18years old. In some embodiments, MCL is pathologically confirmed withdocumentation of either cyclin D1 overexpression and/or presence oft(11:14).

In some embodiments, the CAR T cell intervention comprises T cells whichare expanded from a T cell population depleted of circulating lymphomacells and enriched for CD4+/CD8+ T cells by positive selection ofmononuclear cells from a leukapheresis sample that is activated withanti-CD3 and anti-CD28 antibodies in the presence of IL-2, and thentransduced with a replication-incompetent viral vector containing ananti-CD19 CAR construct. In some embodiments, the CAR construct isFMC63-28Z CAR. The CAR T cell generated using this method may bereferred to as KTE-X19. In some embodiments, the cells are autologous.In some embodiments, the cells are heterologous. In some embodiments,the dose of CAR-positive T cells is 2×10⁶ anti-CD19 CAR T cells/kg. Insome embodiments, the dose of CAR-positive T cells is 1×10⁶ anti-CD19CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is1.6×10⁶ anti-CD19 CAR T cells/kg, 1.8×10⁶ anti-CD19 CAR T cells/kg, or1.9×10⁶ anti-CD19 CAR T cells/kg. In some embodiments, the CD19 CARconstruct contains a CD3ζ T cell activation domain and CD28 signalingdomain.

In some embodiments, the CAR T cells are administered as a singleinfusion on Day 0 following conditioning therapy with 25 mg/m²/day offludarabine on Days −5, −4, and −3 and 900 mg/m²/day of cyclophosphamideon Day −2, after leukapheresis. In some embodiments, the conditioningtherapy comprises 300 mg/m²/day of cyclophosphamide and 30 mg/m²/day offludarabine for 3 days. In some embodiments, the conditioningchemotherapy comprises 30 mg/m²/day of fludarabine and 500 mg/m²/day ofcyclophosphamide on Days −5, −4, and −3. In some embodiments, thepatient may also have received acetaminophen and diphenhydramine oranother H1-antihistamine approximately 30 to 60 minutes prior toinfusion of anti-CD19 CAR T cells. In some embodiments, the patientsreceive one or more additional doses of anti-CD19 CAR T cells.

In some embodiments, the MCL cancer is relapsed/refractory MCL (r/rMCL). In some embodiments, the patient has received one or more priortreatments. In some embodiments, the patient has received 1-5 priortreatments. In some embodiments, the prior treatments may have includedautologous SCT, anti-CD20 antibody, anthracycline- orbendamustine-containing chemotherapy, and/or a Bruton Tyrosine Kinaseinhibitor (BTKi). In some embodiments, the BTKi is ibrutinib (Ibr). Insome embodiments, the BTKi is acalabrutinib (Acala). In someembodiments, the disclosure provides that MCL patients who werepreviously treated with ibrutinib had a more pronounced response toanti-CD19 CAR T cell therapy as compared to patients previously treatedwith acalabrutinib. Accordingly, the disclosure provides a method oftreating r/r MCL with anti-CD19 CAR T cell therapy wherein the patienthas been previously treated with ibrutinib or acalabrutinib and whosecancer is, preferably, relapsed/refractory to the same. In someembodiments, the BTKi is tirabrutinib (ONO-4059), zanubrutinib(BGB-3111), CGI-1746 or spebrutinib (AVL-292, CC-292).

In some embodiments, the disclosure provides that for patients withprior Ibr, Acala, or both, median (range) peak CART cell levels were95.9 (0.4-2589.5), 13.7 (0.2-182.4), or 115.9 (17.2-1753.6),respectively. In some embodiments, ORR/CR rates to anti-CD19 CAR T celltherapy in patients with MCL were 94%/65% in patients with prior Ibr,80%/40% in patients with prior Acala, and 100%/100% in patients withboth BTKis. In some embodiments, the 12-month survival rates in patientswith prior Ibr, Acala, or both were 81%, 80%, or 100%, respectively. Insome embodiments, CAR T cell expansion is associated with ORR/CR rate inpatients previously treated with Ibr and/or Acala. Accordingly, in oneembodiment, the patient is treated with both Ibr and Acala. In oneembodiment, the disclosure provides a method of predicting ORR/CR in anMCL patient previously treated with Ibr and/or Acala by measuring peakCAR T cell levels and comparing them to a reference standard. In oneembodiment, the disclosure provides a method of predicting ongoingresponse based on the measurement of CAR T cell peak levels/baselinetumor burden (CEN and INV). In one embodiment, the higher the ratio, thehigher the likelihood of ongoing response at/by 12 months. In oneembodiment, a ratio between 0.00001 and 0.005 is predictive ofnon-response at/by 12 months. In one embodiment, a ratio between 0.006and 0.3 is predictive of relapse at/by 12 months. In one embodiment, aration between 0.4 and 1 is predictive of ongoing response at/by 12months. In one embodiment, the ratios may be determined by one ofordinary skill in the art from the average populations.

In some embodiments, additional inclusion criteria include those listedin EXAMPLE 2. In some embodiments, additional exclusion criteria includethose listed in EXAMPLE 2.

In some embodiments, the patient may have received bridging therapy(after leukapheresis and before chemotherapy) with dexamethasone (e.g.,20-40 mg or equivalent PO or IV daily for 1-4 days), methylprednisolone,ibrutinib (e.g., 560 mg PO daily), and/or acalabrutinib (e.g, 100 mg POtwice daily) after leukapheresis and completed, for example, in 5 daysor less before conditioning chemotherapy. In some embodiments, suchpatient may have had high disease burden. In some embodiments, thebridging therapy is selected from an immunomodulator, R-CHOP,bendamustine, alkylating agents, and/or platinum-based agents.

In some embodiments, the disclosure provides that all MCL patients whoresponded to CAR T cell infusion achieved T cell expansion, whereas noexpansion was observed in non-responding patients. In some embodiments,response is objective response (complete response+partial response). Thedisclosure provides that CAR T cell levels correlated with ORR in thefirst 28 days, where the area under the curve on days 0 to 28 (AUC₀₋₂₈)and peak levels were >200-fold higher in responders versusnon-responders, suggesting that higher expansion led to better andperhaps deeper response as also indicated by the >80-fold higherpeak/AUC CAR T cell levels in minimal residual disease (MRD, 10⁻⁵sensitivity) negative compared with MRD positive patients (at week 4).Accordingly, the disclosure provides a method of predicting patientresponse and MRD to CAR T cell treatment of MCL comprising measuringpeak/AUC CAR T cell levels and comparing them to a reference standard.In some embodiments, peak CAR T cell expansion is observed between Days8 and 15 after CAR T cell administration. In some embodiments, CAR Tcells levels are measured by qPCR. In some embodiments, the peak CAR Tcell levels, AUC₀₋₂₈, and/or MRD are monitored by next-generationsequencing. In some examples, the CAR T cell numbers are measured incells/microliter of blood. In some examples, the CAR T cell numbers aremeasured by the number of CAR gene copies/μg of host DNA. In someexamples, the CAR T cell numbers are measured as described inKochenderfer J. N et al. J. Clin. Oncol. 2015; 33:540-549. In oneembodiment, CAR T cell levels are measured as described in Locke Fla. etal. Mol Ther. 2017; 25(1):285-295.

In some embodiments, the disclosure provides that there is a differencebetween T cell expansion of responders and nonresponders. In someembodiments, the disclosure provides that the median peak anti-CD19 CART cell level in responders (those with complete remission and partialremission) was 102.4 cells/μL (range: 0.2 to 2589.5 cells/μL; n=51), andin nonresponders was 12.0 cells/μL (range: 0.2 to 1364.0 cells/μL, n=8).In some embodiments, the disclosure provides that the median AUC Day0-28 (AUC₀₋₂₈) in patients with an objective response was 1487.0cells/μL·days (range: 3.8 to 2.77×10⁴ cells/μL·days; n=51) and 169.5cells/μL·days in nonresponders (range: 1.8 to 1.17 10×10⁴ cells/μL·days;n=8). The median peak (24.7 cells/μL) anti-CD19 CAR T cell (peak: andAUC₀₋₂₈ levels (360.4 cells/μL·days) in patients (n=18) who receivedneither corticosteroids nor tocilizumab was similar to those of patients(n=2) who received only corticosteroids (peak: 24.2 cells/μL; AUC₀₋₂₈:367.8 cells/μL·days). In the patients who received only tocilizumab(n=10), the mean peak anti-CD19 CAR T cells was 86.5 cells/μL andAUC₀₋₂₈ was 1188.9 cells/μL·days. In the patients who received bothcorticosteroids and tocilizumab (n=37), the mean peak was 167.2 cells/μLand AUC₀₋₂₈ was 1996.0 cells/μL·days. The median peak anti-CD19 CART-cell values were 74.1 cells/μL in patients ≥65 years of age (n=39) and112.5 cells/μL in patients ≤65 years of age (n=28). Median anti-CD19 CART-cell AUC₀₋₂₈ values were 876.5 cells/μL·day in patients ≥65 years ofage and 1640.2 cells/μL·day in patients <65 years of age. Gender had nosignificant impact on AUC₀₋₂₈ and C_(max) of anti-CD19 CAR T cells.Accordingly, the disclosure provides a method of predicting response inMCL comprising measuring T cell expansion after anti-CD19 CAR Ttreatment and comparing the level to a reference standard.

In some embodiments, the disclosure provides that CAR T cell expansionwas greater in MCL patients with grade ≥3 than in those with grade ≤3CRS and NE events. Accordingly, the disclosure provides a method ofpredicting grade ≥3 CRS and NE events comprising measuring CAR T cellexpansion after CAR T cell treatment and comparing the levels to areference value, wherein the higher the CAR T cell expansion, the higherthe chance for grade ≥3 CRS and NE events.

In some embodiments, the cytokine levels are measured by and are proteinor mRNA levels (which ones). In some embodiments, the cytokine levelsare measured as described in Locke F L et al. Mol Ther. 2017;25(1):285-295.

In some embodiments, the disclosure provides that serum GM-CSF and IL-6peak levels (reached about 8 days post CAR T cell administration) werepositively associated with grade ≥3 CRS and grade ≥3 NE in MCL patients.Accordingly, the disclosure provides a method of predicting grade ≥3 CRSand grade ≥3 NE comprising measuring the peak levels of GM-CSF and IL-6post-CAR T cell administration and comparing them to a reference level,wherein the higher the peak level of these cytokines, the higher thechance for grade ≥3 CRS and NE.

In some embodiments, the disclosure provides that serum ferritin waspositively associated with grade ≥3 CRS in MCL patients. Accordingly,the disclosure provides a method of predicting grade ≥3 CRS comprisingmeasuring the peak levels of serum ferritin post-CAR T celladministration and comparing them to a reference level, wherein thehigher the peak level of ferritin, the higher the chance for grade ≥3CRS.

In some embodiments, the disclosure provides that serum IL-2 andIFN-gamma were positively associated with grade ≥3 NE in MCL patients.Accordingly, the disclosure provides a method of predicting grade ≥3 CRScomprising measuring the peak levels of serum IL-2 and IFN-gammapost-CAR T cell administration and comparing them to a reference level,wherein the higher the peak level of IL-2 and IFN-gamma, the higher thechance for grade ≥3 NE.

In some embodiments, the disclosure provides that cerebrospinal fluidlevels of C-reactive protein, ferritin, IL-6, IL-8, and vascular celladhesion molecule (VCAM) were positively associated with grade ≥3 NE inMCL patients. Accordingly, the disclosure provides a method ofpredicting grade ≥3 CRS comprising measuring the cerebrospinal fluidlevels of C-reactive protein, ferritin, IL-6, IL-8, and/or vascular celladhesion molecule (VCAM) post-CAR T cell administration and comparingthem to a reference level, wherein the higher the cerebrospinal fluidlevels of C-reactive protein, ferritin, IL-6, IL-8, and/or vascular celladhesion molecule (VCAM), the higher the chance for grade ≥3 NE. In someembodiments, one or more adverse events were managed according to Table13 and/or Table 14.

In some embodiments, the disclosure provides that peak serum levels ofcytokines associated positively with Grade ≥3 CRS included IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzymeA, granzyme B, and perforin. In some embodiments, the disclosureprovides that peak serum levels of cytokines associated with Grade ≥3 NEincluded IL-2, IL-1 Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10,MCP-4, MIP-1b, and granzyme B. In some embodiments, the disclosureprovides that cytokines associated with both Grade ≥3 CRS and NEincluded IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B. In someembodiments, cytokine serum levels peak within 7 days of CAR T celladministration. Accordingly, the disclosure provides a method ofpredicting grade ≥3 CRS post-CAR T cell administration comprisingmeasuring peak serum levels of IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF,ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and/orperforin after anti-CD19 CAR T treatment and comparing the levels to areference standard. Accordingly, the disclosure also provides a methodof predicting grade ≥3 CRS and grade ≥3 NE in MCL comprising measuringpeak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme Bafter anti-CD19 CAR T treatment and comparing the levels to a referencestandard.

In some embodiments, the disclosure provides that there was a trend forincreased proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2Rα,sPD-L1 and VCAM-1) peak cytokine levels in patients with MCL withmutated TP53 vs wild-type TP53. Accordingly, in some embodiments, thedisclosure provides a method of improving response to CAR T celltreatment in MCL comprising manipulating the levels of proliferativeand/or inflammatory cytokines after CAR T cell administration.

In some embodiments, the disclosure provides that for patients that wereMRD negative at one month post CAR T cell administration, there was anincrease in peak levels of IFN-gamma and IL-6, and a trend towardsincreased IL-2, relative to patients that were MRD positive at onemonth. Accordingly, the disclosure provides a method of predictingwhether a patient is MRD negative in MCL comprising measuring peak serumlevels of IFN-gamma, IL-6, and/or IL-2 after anti-CD19 CAR T treatmentand comparing the level to a reference standard.

In some embodiments, the disclosure provides that the T cell productphenotype varied among types of MCL. In some embodiments, the disclosureprovides that, in the manufactured anti-CD19 CAR T product, median(range) CD4+/CD8+ T cell ratios for patients with classical, blastoid,or pleomorphic MCL were 0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0),respectively. Product T cell phenotypes (median [range]) included lessdifferentiated CCR7+ T cells (classical 40.0% [2.6-88.8]; blastoid 35.3%[14.3-73.4]; pleomorphic 80.8% [57.3-88.8]) and effector and effectormemory CCR7− T cells (classical 59.9% [11.1-97.4]; blastoid 64.8%[26.6-85.7]; pleomorphic 19.2% [11.1-42.7]). In some embodiments, thedisclosure provides that the 12-mo survival rates in patients withclassical, blastoid, or pleomorphic MCL were 86.7%, 67.9%, or 100%,respectively. Accordingly, the disclosure provides a method of improvingtreatment of classical, blastoid, or pleomorphic MCL by manipulating theT cell product phenotype administered to the patient.

Exemplary Treatment of B Cell ALL

B-ALL cells typically express CD19, and CAR T-cell therapies targetingCD19 are a treatment approach in R/R B-ALL. Pehlivan K. C., et al. CurrHematol Malig Rep. 2018; 13(5):396-406 An anti-CD19 CAR T-cell therapycontaining a CD3ζ and CD28 co-stimulatory domain developed at theNational Cancer Institute (Kochenderfer J N et al. J Immunother. 2009;32(7):689-702; Kochenderfer J N et al. Blood. 2010; 116(19):3875-3886)showed an overall remission rate of 70% after a median 10-monthfollow-up in a phase 1 trial in children and adults ≤30 years of agewith R/R B-ALL. Lee D W et al. Lancet. 2015; 385(9967):517-528. Asimilar CAR construct evaluated in a phase 1 trial in adults with R/RB-ALL provided an 83% complete remission (CR) rate and median 12.9-monthOS at a median 29-months follow-up. Park J H et al. N Engl J Med. 2018;378(5):449-459. In these studies, the CAR T cells were prepared fromleukapheresis samples that were not enriched for CD4+/CD8+ T cells.

In some embodiments, the disclosure is directed to a T cells productwhereby the T cells are expanded from a T cell population depleted ofcirculating lymphoma cells and enriched for CD4+/CD8+ T cells bypositive selection of mononuclear cells from a leukapheresis sample thatis activated with anti-CD3 and anti-CD28 antibodies in the presence ofIL-2, and then transduced with a replication-incompetent viral vectorcontaining an anti-CD19 CAR construct. In some embodiments, such T cellproduct may be used to treat ALL, CLL, AML. In some embodiments, the CARconstruct is FMC63-28Z CAR. In some embodiments, the cells areautologous. In some embodiments, the cells are heterologous. In someembodiments, the dose of CAR-positive T cells is 2×10⁶ anti-CD19 CAR Tcells/kg. In some embodiments, the dose of CAR-positive T cells is 1×10⁶anti-CD19 CAR T cells/kg. In some embodiments, the dose of CAR-positiveT cells is 1.6×10⁶ anti-CD19 CAR T cells/kg, 1.8×10⁶ anti-CD19 CAR Tcells/kg, or 1.9×10⁶ anti-CD19 CAR T cells/kg. In some embodiments, theCD19 CAR construct contains a CD3ζ T cell activation domain and CD28signaling domain. In some embodiments, the T cell product is KTE-X19. Insome embodiments, the disclosure provides that the anti-CAR T cellproduct prepared as described in the preceding paragraph may be used inB cell ALL and B cell NHL. In one embodiment, the T cell product has thecharacteristics of the products of Table 23. In some embodiments, theproduct characteristics may be selected from percentage of T cells ofspecific subsets (naïve, central memory, effector, and effector memory),percentage of CD4+ cells, percentage of CD8+ cells and CD4/CD8 ratio. Insome embodiments, the product characteristic is the level of IFNγproduction in co-culture (pg/mL) with target CD19-expressing cancercells (e.g. Toledo) cells mixed in a 1:1 ratio with the anti-CD19 CAR Tproduct cells. In one embodiment, IFNγ may be measured in cell culturemedia 24 h post-incubation using a qualified ELISA. In some embodiments,one or more of these product characteristics is superior than those ofanti-CAR T cells prepared from leukapheresis without CD4+/CD8+ positivecell enrichment. In some embodiments, the superior productcharacteristic may be selected from increased percentage of cells withnaïve phenotype (CD45RA+ CCR7+), decreased percentage of cells withdifferentiated phenotype (CCR7−), decreased level of IFNγ-producingcells, and increased level of CD8+ cells. In some embodiments, theanti-CD19 T cell product comprises T_(CM), central memory T cells(CD45RA-CCR7+); TEFF, effector T cells (CD45RA+ CCR7−); TEM, effectormemory T cells (CD45RA-CCR7−); and/or TN, naïve-like T cells (CD45RA+CCR7+). In some embodiments, the product comprises TN naïve-like T cellsmeans T cells that are CD45RA+ CCR7+ and comprises stem-like memorycells. In some embodiments, the T cell product is KTE-X19. In someembodiments, KTE-X19 has ≥190 pg/mL IFN-γ production. In certainembodiment, KTE-X19 has ≥90% of CD3+ cells. In some other embodiments,the percentage of NK cells in KTE-X19 is 0.1% (range 0.0%-2.8%). In someadditional embodiments, the percentage of CD3⁻ cellular impurities inKTE-X19 is 0.5% (range 0.3%-3.9%).

In some embodiments, the cancer is relapsed/refractory B cell ALL. Insome embodiments, the patient is ≤21 years-old. In some embodiments, thepatient is ≤21 years-old, weighs ≥10 kg, and has B cell ALL that isprimary refractory, relapsed within 18 months of first diagnosis, R/Rafter ≥2 lines of systemic therapy, or R/R after allogeneic stem celltransplantation at least 100 days prior to enrollment. In oneembodiment, the cancer is indolent lymphoma or leukemia. In oneembodiment, the cancer is an aggressive B-cell lymphoma, which includemany types, subtypes and variants of diffuse large B-cell lymphoma(DLBCL), Burkitt lymphoma (BL), mantle cell lymphoma and its blastoidvariant, and B lymphoblastic lymphoma. DLBCL may be DLBCL NOS,T-cell/histiocyte-rich large B-cell lymphoma, Primary DLBCL of the CNS,Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly.Other lymphomas of large B cells include Primary mediastinal (thymic)LBCL, DLBCL associated with chronic inflammation, Lymphomatoidgranulomatosis, ALK-positive LBCL, Plasmablastic lymphoma, Large B-celllymphoma arising in HHV8-associated multicentric Castleman disease, andPrimary effusion lymphoma. Other types of lymphomas include B-celllymphoma, unclassifiable, with features intermediate between DLBCL, andBurkitt's lymphoma and B-cell lymphoma, unclassifiable, with featuresintermediate between DLBCL and classical Hodgkin lymphoma, Splenicmarginal zone B-cell lymphoma, Extranodal marginal zone B-cell lymphomaof MALT, Nodal marginal zone B-cell lymphoma, Hairy cell leukemia,Lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), and Primaryeffusion lymphoma. The cancer may be at any stage, from stage 1 throughstage 4.

ALL is a common childhood malignancy, constituting approximately 80% ofchildhood leukemias and approximately 25% of all childhood cancers.Approximately 20% of pediatric patients do not achieve long-termremission after initial therapy, with a 5-year OS rate of approximately55%. Hunger S P, et al. N Engl J Med. 2015; 373:1541-1552; Sun W, et al.Leukemia. 2018; 32:2316-2325; Rheingold S R, et al. J Clin Oncol. 2019;37(suppl, abstr):10008 and Oskarsson T, et al. Haematologica. 2016;101:68-76. Outcomes are poor for patients who relapse early or haveprimary refractory disease after initial treatment; patients with R/Rdisease after stem cell transplantation; and multiply relapsed patients.Sun W, et al. Leukemia. 2018; 32:2316-2325; Rheingold S R, et al. J ClinOncol. 2019; 37(suppl, abstr):10008; Oskarsson T, et al. Haematologica.2016; 101:68-76; Nguyen K, et al. Leukemia. 2008; 22:2142-2150; CrottaA, et al. Curr Med Res Opin. 2018; 34:435-440; Schrappe M, et al. N EnglJ Med. 2012; 366:1371-1381. Patients who relapse within 18 months ofinitial diagnosis generally have a 5-year OS rate of 21%-28%. RheingoldS R, et al. J Clin Oncol. 2019; 37(suppl, abstr):10008; Nguyen K, et al.Leukemia. 2008; 22:2142-2150. The likelihood of achieving remission andthe duration of EFS decrease with each subsequent line of salvagetherapy. Sun W, et al. Leukemia. 2018; 32:2316-2325. Outcomes remainpoor in pediatric and adolescent patients with R/R ALL after treatmentwith the novel therapies blinatumomab and inotuzumab ozogamicin, with a1-year OS rate of approximately 36%, highlighting the need for moreeffective therapeutic options. von Stackelberg A, et al. J Clin Oncol.2016; 34:4381-4389. 10; Bhojwani D, et al. Leukemia. 2019; 33:884-892.

In some embodiments, the cancer is B cell NHL and key enrollmentcriteria include age ≤18 years, weight ≥10 kg, histologically confirmeddiffuse large B cell lymphoma not otherwise specified (DLBCL NOS),primary mediastinal large B cell lymphoma, Burkitt lymphoma (BL),Burkitt-like lymphoma or unclassified B cell lymphomas intermediatebetween DLBCL and BL, with ≥1 measurable lesion. In one embodiment, forNHL treatment, the disease may have been primary refractory, R/R after≥2 lines of systemic therapy, or R/R after autologous or allogeneic stemcell transplantation ≥100 days prior to enrollment. Patients with acutegraft-versus-host disease or chronic graft-versus-host disease requiringtreatment within 4 weeks of enrollment may not be eligible.

In some embodiments, these B cell ALL and/or the B cell NHL patientsreceive conditioning chemotherapy with fludarabine 25 mg/m²/day on Days−4, −3, and −2 and cyclophosphamide 900 mg/m²/day on Day −2 followed bya single infusion of CD4+/CD8+-enriched anti-CD19 CAR T cells (preparedas described immediately above) at a target dose of 1×10⁶ anti-CD19 CART cells/kg on Day 0.

In some embodiments, the disclosure provides the use of CD4+/CD8+enriched/cancer cell depleted anti-CD19 CAR T cells to successfullytreat B cell ALL, where the patient is ≥18 years of age with R/R B cellALL, defined as refractory to first-line therapy (i.e., primaryrefractory), relapse ≤12 months after first remission, relapsed orrefractory after ≥2 prior lines of systemic therapy, or relapsed afterallogeneic stem cell transplant (SCT). In some embodiments, patientswere required to have ≥5% bone marrow blasts, an Eastern CooperativeOncology Group performance status of 0 or 1, and adequate renal,hepatic, and cardiac function. For patients who received priorblinatumomab, leukemic blasts with CD19 expression ≥90% was required.Patients with Philadelphia chromosome-positive (Ph+) disease,concomitant extramedullary disease, central nervous system (CNS)-2disease (cerebrospinal fluid [CSF] blast cells with <5 white bloodcells/mm³) without neurological changes and patients with Down syndromewere eligible. CNS-3 disease (CSF blast cells with ≥5 white bloodcells/mm³) independent of neurologic changes and a history of CNSdisorder were exclusions. In some embodiments, additional inclusion andexclusion criteria are described in EXAMPLE 9.

In some embodiments, the patient may have a cancer that is primaryrefractory. In some embodiments, the patient may have a cancer that hasrelapsed after SCT. In some embodiments, the patient may have receivedprior blinatumomab, which may have been the last therapy used prior toanti-CD19 CAR T cell therapy. In some embodiments, the patient baselinecharacteristics are those of any one of the patients described in Table18.

In some embodiments, these B cell ALL patients are administered 2×10⁶,1×10⁶, or 0.5×10⁶ CAR T cells/kg. In some embodiments, the 0.5×10⁶ CAR Tcells/kg are administered in a formulation with a total volume of 40 mL.In another embodiment, the 0.5×10⁶ CAR T cells/kg are administered in aformulation with a total volume of 68 mL. In some embodiments, the CAR Tcell product is formulated in a total volume of 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500,700, 800, 900, or 1000 mL. In some embodiments, the 40 mL formulation isintended to maintain cell density and cell viability during thefreezing/thawing process. In some embodiments, the treatment isassociated with adverse events. In some embodiments, one or more adverseevents is managed in accordance with any one of Tables 13, 14, 16, orcombinations thereof. In some embodiments, the one or more adverseevents is managed in accordance with the Original Management Guidelinesof Table 16. In some embodiments, one or more adverse events is managedin accordance with the Revised Management Guidelines of Table 16. Insome embodiments, vasopressors may be administered to treat CRS. In someembodiments, signs or symptoms associated with CRS, include fever,chills, fatigue, tachycardia, nausea, hypoxia, and hypotension. In someembodiments, signs or symptoms associated with neurologic events,including encephalopathy, seizures, changes in level of consciousness,speech disorders, tremors, and confusion.

In some embodiments, the patient may have a high disease burden atbaseline, which is defined as having (≥25% leukemic blasts in bonemarrow or ≥1,000 blasts/mm³ in peripheral circulation by local review.In some embodiments, the patients may receive bridging chemotherapyafter leukapheresis and before conditioning chemotherapy. In someembodiments, the bridging chemotherapy follows one of the predefinedbridging chemotherapy regimens of Table 17.

In some embodiments, the conditioning chemotherapy/lymphodepletingregimen is administered after ≥7 days or 5 half-lives (if shorter)washout from bridging chemotherapy. In some embodiments, theconditioning chemotherapy/lymphodepleting regimen consists offludarabine intravenous (IV) 25 mg/m²/day on days −4, −3, and −2, andcyclophosphamide IV 900 mg/m²/day on day −2. On day 0, a single infusionof anti-CD19 CAR T cells may be administered. In some embodiments,additional infusions of anti-CD19 CAR T cells may be administered at alater time. In some embodiments, patients achieving complete response tothe first infusion may receive a second infusion of anti-CD19 CAR Tcells, if progressing following >3 months of remission, provided CD19expression has been retained and neutralizing antibodies against the CARare not suspected.

In some embodiments, droplet digital polymerase chain reaction may beused to measure the presence, expansion, and persistence of transducedanti-CD19 CAR+ T cells in the blood. In some embodiments, the procedureis as described in Locke F. L. et al. Mol Ther. 2017; 25(1):285-295. Insome embodiments, the disclosure provides a method of treatment wherebythe CAR T cell levels are as described in Table 22. In some embodiments,the disclosure provides that CAR T cells may be undetectable at relapse.Median peak CAR T-cell levels may be highest with 1×10⁶ CAR T cells/kgand may be similar between patients who received original vs. revised AEmanagement. In some embodiments, patients achieving CR/CRi had greatermedian peak expansion than non-responders, as did patients withundetectable vs. detectable MRD. Higher median peak expansion was alsoobserved in patients with grade ≥3 NE vs. those with grade ≤2 NE. Somepatients who relapse may have detectable CD19-positive cells at relapseor may have no detectable CD19-postive cells. In some embodiments,undetectable MRD, defined as <1 leukemia cell per 10,000 viable cells,may be assessed using flow cytometry (NeoGenomics, Fort Myers, Fla.) asper the methods described in Borowitz M J, Wood B L, Devidas M, et al.Blood. 2015; 126(8):964-971; Bruggemann M. et al. Blood Adv. 2017;1(25):2456-2466; or Gupta S. et al. Leukemia. 2018; 32(6):1370-1379.

In some embodiments, the disclosure provides that peak levels of somecytokines, chemokines, and pro-inflammatory markers occurred by day 7.In some embodiments, some of these trended higher in patients dosed with2×10⁶ compared with 1×10⁶ CAR T cells/kg (IL-15, CRP, SAA, CXCL10,IFNγ), or lower in those with revised AE management vs those withoriginal AE management (IL-6, Ferritin, IL-1RA, IFNγ, IL-8, CXCL10,MCP-1). In some embodiments, the levels of these proteins/biomarkerschange as described in FIG. 9 ; FIG. 10 ; and FIG. 11 ). Accordingly, insome embodiments, the disclosure provides methods for using theseprotein levels as biomarkers for Grade ≥3 and/or Grade 0-2 CRS. In someembodiments, the disclosure provides methods for using these proteinlevels as biomarkers for Grade ≥3 and/or Grade 0-2 CRS, according totheir values in FIG. 11 .

In some embodiments, the disclosure provides that peak IL-15 serumlevels are lower in patients with grade ≥3 CRS. In some embodiments, thedisclosure provides that median peak levels of several pro-inflammatorymarkers trended higher in patients with grade ≥3 CRS and those withgrade ≥3 NE (IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, Granzyme B, asdescribed in FIG. 11 . Accordingly, in some embodiments, the disclosureprovides a method for predicting whether a patient is going to havegrade ≥3 CRS by measuring the peak levels of serum IL-15 and comparingto a reference standard. In some embodiments, the disclosure provides amethod for predicting whether a patient is going to have grade ≥3 CRSand/or grade ≥3 NE by measuring the peak levels of IFNγ, IL-8, GM-CSF,IL-1RA, CXCL10, MCP-1, and/or Granzyme B and comparing to a referencestandard. In some embodiments, the disclosure provides a method forimproving anti-CD19 CAR T cell therapy by administering agents thatdecrease the levels of one or more of these biomarkers.

The reference levels/standards may be established by any method known byone of ordinary skill in the art. They serve to identify thresholds orgroups of values (e.g., quartiles) from which a comparison may be madeto determine in which group, or above or below which threshold does themeasured value (cytokine level, CAR T cell number, etc.) for eachsubject fall. These groups are established from comparisons of differentpopulations chosen as is typical in the art. Depending on where themeasured value falls, one can predict a number of treatmentcharacteristics such as objective response, CRS grade, NE grade, and thelike.

In certain embodiments, the cancer may be selected from a tumor derivedfrom acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),adenoid cystic carcinoma, adrenocortical, carcinoma, AIDS-relatedcancers, anal cancer, appendix cancer, astrocytomas, atypicalteratoid/rhabdoid tumor, central nervous system, B-cell leukemia,lymphoma or other B cell malignancies, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, osteosarcoma and malignant fibroushistiocytoma, brain stem glioma, brain tumors, breast cancer, bronchialtumors, burkitt lymphoma, carcinoid tumors, central nervous systemcancers, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), chronic myeloproliferativedisorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneoust-cell lymphoma, embryonal tumors, central nervous system, endometrialcancer, ependymoblastoma, ependymoma, esophageal cancer,esthesioneuroblastoma, ewing sarcoma family of tumors extracranial germcell tumor, extragonadal germ cell tumor extrahepatic bile duct cancer,eye cancer fibrous histiocytoma of bone, malignant, and osteosarcoma,gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumors (GIST), soft tissue sarcoma, germcell tumor, gestational trophoblastic tumor, glioma, hairy cellleukemia, head and neck cancer, heart cancer, hepatocellular (liver)cancer, histiocytosis, hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, islet cell tumors (endocrine pancreas), kaposisarcoma, kidney cancer, langerhans cell histiocytosis, laryngeal cancer,leukemia, lip and oral cavity cancer, liver cancer (primary), lobularcarcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, malebreast cancer, malignant fibrous histiocytoma of bone and osteosarcoma,medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma,mesothelioma, metastatic squamous neck cancer with occult primarymidline tract carcinoma involving NUT gene, mouth cancer, multipleendocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm,mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia,chronic (CML), Myeloid leukemia, acute (AML), myeloma, multiple,myeloproliferative disorders, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-smallcell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer,osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer,pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineal parenchymal tumors of intermediatedifferentiation, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiplemyeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primarycentral nervous system (CNS) lymphoma, prostate cancer, rectal cancer,renal cell (kidney) cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma, sézary syndrome, small cell lung cancer, small intestinecancer, soft tissue sarcoma, squamous cell carcinoma, squamous neckcancer, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, t-cell lymphoma, cutaneous, testicular cancer,throat cancer, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter, trophoblastictumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer,uterine sarcoma, vaginal cancer, vulvar cancer, Waldenströmmacroglobulinemia, Wilms Tumor. In certain embodiments, the cancer istreated with KTE-X19.

In one embodiment, the method may be used to treat a tumor, wherein thetumor is a lymphoma or a leukemia. Lymphoma and leukemia are cancers ofthe blood that specifically affect lymphocytes. All leukocytes in theblood originate from a single type of multipotent hematopoietic stemcell found in the bone marrow. This stem cell produces both myeloidprogenitor cells and lymphoid progenitor cell, which then give rise tothe various types of leukocytes found in the body. Leukocytes arisingfrom the myeloid progenitor cells include T lymphocytes (T cells), Blymphocytes (B cells), natural killer cells, and plasma cells.Leukocytes arising from the lymphoid progenitor cells includemegakaryocytes, mast cells, basophils, neutrophils, eosinophils,monocytes, and macrophages. Lymphomas and leukemias may affect one ormore of these cell types in a patient. In certain embodiments, the tumoris treated with KTE-X19.

In general, lymphomas may be divided into at least two sub-groups:Hodgkin lymphoma and non-Hodgkin lymphoma. Non-Hodgkin Lymphoma (NHL) isa heterogeneous group of cancers originating in B lymphocytes, Tlymphocytes or natural killer cells. In the United States, B celllymphomas represent 80-85% of cases reported. In 2013 approximately69,740 new cases of NHL and over 19,000 deaths related to the diseasewere estimated to occur. Non-Hodgkin lymphoma is the most prevalenthematological malignancy and is the seventh leading site of new cancersamong men and women and account for 4% of all new cancer cases and 3% ofdeaths related to cancer. In certain embodiments, the lymphoma istreated with KTE-X19.

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of NHL,accounting for approximately 30% of NHL cases. There are approximately22,000 new diagnoses of DLBCL in the United States each year. It isclassified as an aggressive lymphoma with the majority of patients curedwith conventional chemotherapy (NCCN guidelines NHL 2014). First linetherapy for DLBCL typically includes an anthracycline-containing regimenwith rituximab, such as R-CHOP (rituximab, cyclophosphamide,doxorubicin, vincristine, and prednisone), which has an objectiveresponse rate of about 80% and a complete response rate of about 50%,with about one-third of patients have refractory disease to initialtherapy or relapse after R-CHOP. For those patients who relapse afterresponse to first line therapy, approximately 40-60% of patients mayachieve a second response with additional chemotherapy. The standard ofcare for second-line therapy for autologous stem cell transplant (ASCT)eligible patients includes rituximab and combination chemotherapy suchas R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP(rituximab, dexamethasone, cytarabine, and cisplatin), which each havean objective response rate of about 63% and a complete response rate ofabout 26%. Patients who respond to second line therapy and who areconsidered fit enough for transplant receive consolidation withhigh-dose chemotherapy and ASCT, which is curative in about half oftransplanted patients Patients who failed ASCT have a very poorprognosis and no curative options. Primary mediastinal large B celllymphoma (PMBCL) has distinct clinical, pathological, and molecularcharacteristics compared to DLBCL. PMBCL is thought to arise from thymic(medullary) B cells and represents approximately 3% of patientsdiagnosed with DLBCL. PMBCL is typically identified in the younger adultpopulation in the fourth decade of life with a slight femalepredominance. Gene expression profiling suggests deregulated pathways inPMBCL overlap with Hodgkin lymphoma. Initial therapy of PMBCL generallyincludes anthracycline-containing regimens with rituximab, such asinfusional dose-adjusted etoposide, doxorubicin, and cyclophosphamidewith vincristine, prednisone, and rituximab (DA-EPOCH-R), with orwithout involved field radiotherapy. Follicular lymphoma (FL), a B celllymphoma, is the most common indolent (slow-growing) form of NHL,accounting for approximately 20% to 30% of all NHLs. Some patients withFL will transform (TFL) histologically to DLBCL which is more aggressiveand associated with a poor outcome. Histological transformation to DLBCLoccurs at an annual rate of approximately 3% for 15 years with the riskof transformation continuing to drop in subsequent years. The biologicmechanism of histologic transformation is unknown. Initial treatment ofTFL is influenced by prior therapies for follicular lymphoma butgenerally includes anthracycline-containing regimens with rituximab toeliminate the aggressive component of the disease. Treatment options forrelapsed/refractory PMBCL and TFL are similar to those in DLBCL. Giventhe low prevalence of these diseases, no large prospective randomizedstudies in these patient populations have been conducted. Patients withchemotherapy refractory disease have a similar or worse prognosis tothose with refractory DLBCL. As an example, subjects who haverefractory, aggressive NHL (e. g., DLBCL, PMBCL and TFL) have a majorunmet medical need and further research with novel treatments arewarranted in these populations. In certain embodiments, the DLBCL istreated with KTE-X19.

The CAR T cell treatment of the disclosure may be administered as afirst line of treatment or a second or later line of treatment. In someembodiments, the CAR T cell treatment is administered as a third line,fourth line, fifth line and so on and so forth. The lines of priortherapy may be any prior anti-cancer therapy, including, but not limitedto Bruton Tyrosine Kinase inhibitor (BTKi), check-point inhibitors(e.g., anti-PD1 antibodies, pembrolizumab (Keytruda), Cemiplimab(Libtayo), nivolumab (Opdivo); anti-PD-L1 antibodies, Atezolizumab(Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi); anti-CTLA-4antibodies, Ipilimumab (Yervoy)), anti-CD19 antibodies (e.g.blinatumomab), anti-CD52 antibodies (e.g. alentuzumab); allogeneic stemcell transplantation, anti-CD20 antibodies (e.g., rituximab), systemicchemotherapy, rituximab, anthracycline, ofatumumab, and combinationthereof. The prior therapies may also be used in combination with theCD19 CAR T therapies of the application. In one aspect, the eligiblepatients may have refractory disease to the most recent therapy orrelapse within 1 year after autologous hematopoietic stem celltransplantation (HSCT/ASCT). The CAR T cell treatment may beadministered to patients that have or suspect to have cancers that arerefractory and/or that relapsed to one or more lines of previoustherapy. The cancer may be refractory to first-line therapy (i.e.,primary refractory) or refractory to one or more lines of therapy. Thecancer may have relapsed at twelve months after first remission,relapsed or refractory after two or more lines of prior therapy, orrelapsed after HSCT/ASCT. In some embodiments, the cancer is refractoryto ibrutinib or acalabrutinib. In some embodiments, the cancer is NHL,and the disease must have been primary refractory, R/R after two or morelines of systemic therapy, or R/R after autologous or allogeneic stemcell transplantation ≥100 days prior to enrollment in CAR T cell therapyand off immunosuppressive medications for ≥4 weeks. In certainembodiments, the CAR T cell therapy is KTE-X19.

Accordingly, the method may be used to treat a lymphoma or a leukemia,wherein the lymphoma or leukemia is a B cell malignancy. Examples of Bcell malignancies include, but are not limited to, Non-Hodgkin'sLymphomas (NHL), Small lymphocytic lymphoma (SLL/CLL), Mantle celllymphoma (MCL), FL, Marginal zone lymphoma (MZL), Extranodal (MALTlymphoma), Nodal (Monocytoid B-cell lymphoma), Splenic, Diffuse largecell lymphoma, B cell chronic lymphocytic leukemia/lymphoma, Burkitt'slymphoma, and Lymphoblastic lymphoma. In some aspects, the lymphoma orleukemia is selected from B-cell chronic lymphocytic leukemia/small celllymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma(e.g., Waldenström macroglobulinemia), splenic marginal zone lymphoma,hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma(i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone Bcell lymphoma (e.g., MALT lymphoma), nodal marginal zone B celllymphoma, follicular lymphoma (FL), transformed follicular lymphoma(TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma,diffuse large B cell lymphoma (DLBCL), Epstein-Ban virus-positive DLBCL,lymphomatoid granulomatosis, primary mediastinal (thymic) large B-celllymphoma (PMBCL), Intravascular large B-cell lymphoma, ALK+ large B-celllymphoma, plasmablastic lymphoma, primary effusion lymphoma, largeB-cell lymphoma arising in HHV8-associated multicentric Castleman'sdisease, Burkitt lymphoma/leukemia, T-cell prolymphocytic leukemia,T-cell large granular lymphocyte leukemia, aggressive NK cell leukemia,adult T-cell leukemia/lymphoma, extranodal NK/T-cell lymphoma,enteropathy-associated T-cell lymphoma, Hepatosplenic T-cell lymphoma,blastic NK cell lymphoma, Mycosis fungoides/Sezary syndrome, Primarycutaneous anaplastic large cell lymphoma, Lymphomatoid papulosis,Peripheral T-cell lymphoma, Angioimmunoblastic T cell lymphoma,Anaplastic large cell lymphoma, B-lymphoblastic leukemia/lymphoma,B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities,T-lymphoblastic leukemia/lymphoma, and Hodgkin lymphoma. In some aspect,the cancer is refractory to one or more prior treatments, and/or thecancer has relapsed after one or more prior treatments. In certainembodiments, the leukemia or lymphoma is treated with KTE-X19.

In one embodiment, the cancer is selected from follicular lymphoma,transformed follicular lymphoma, diffuse large B cell lymphoma, andprimary mediastinal (thymic) large B-cell lymphoma. In anotherembodiment, the cancer is diffuse large B cell lymphoma. In someembodiment, the cancer is refractory to or the cancer has relapsedfollowing one or more of chemotherapy, radiotherapy, immunotherapy(including a T cell therapy and/or treatment with an antibody orantibody-drug conjugate), an autologous stem cell transplant, or anycombination thereof. In one embodiment, the cancer is refractory diffuselarge B cell lymphoma. In certain embodiments, the cancer is treatedwith KTE-X19.

In some embodiments, the CAR T cell treatment is KTE-X19 and the canceris selected from MCL, ALL, CLL, and SLL. In some embodiments, the CAR Tcell treatment is KTE-X19 and the cancer is NHL. In some embodiments,the cancer is selected from diffuse large B cell lymphoma not otherwisespecified (DLBCL NOS), primary mediastinal large B cell lymphoma,Burkitt lymphoma (BL), Burkitt-like lymphoma or unclassified B celllymphomas intermediate between DLBCL and BL. In some embodiments, thecancer is relapsed/refractory. In some embodiments, the KTE-X19treatment is administered as first line, second line, or after 1 or moreprior lines of therapy. In some embodiments, the patient is a pediatricpatient, an adolescent patient, an adult patient, less than 65 yearsold, more than 65 years old, or any other age group.

In some embodiment, compositions comprising immune cells disclosedherein may be administered in conjunction with any number of additionaltherapeutic agents. In one embodiment, the additional therapeutic agentis administered concurrently with the T cell therapy. In one embodiment,the additional therapeutic agent is administered prior to, during,and/or after T cell therapy. In one embodiment, the one or moreadditional therapeutic agents is administered prophylactically. In oneaspect, the compositions comprising the immune cells are administered inconjunction with agents for management of adverse events (many of whichare described elsewhere in this application, including the Examplessection). These agents may manage one or more of the signs and symptomsof adverse reactions, such as fever, hypotension, tachycardia, hypoxia,and chills, include cardiac arrhythmias (including atrial fibrillationand ventricular tachycardia), cardiac arrest, cardiac failure, renalinsufficiency, capillary leak syndrome, hypotension, hypoxia, organtoxicity, hemophagocytic lymphohistiocytosis/macrophage activationsyndrome (HLH/MAS), seizure, encephalopathy, headache, tremor,dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrileneutropenia, thrombocytopenia, neutropenia, and anemia.

Examples of such agents include, without limitation, tocilizumab,steroids (e.g., methylprednisolone), rabbit anti-thymocyte globulin. Insome aspect, Vancomycin and aztreonam (each 1 gm IV twice daily) may beadministered for non-neutropenic fever. In some aspects, the methodfurther comprises administering a non-sedating, anti-seizure medicinefor seizure prophylaxis; administering at least one of erythropoietin,darbepoetin alfa, platelet transfusion, filgrastim, or pegfilgrastim;and/or administering tocilizumab, siltuximab. In one aspect, the agentis a CSF family member such as GM-CSF (Granulocyte-macrophagecolony-stimulating factor, also known as CSF2). GM-CSF may be producedby a number of haemopoietic and nonhaemopoietic cell types uponstimulation, and it may activate/‘prime’ myeloid populations to produceinflammatory mediators, such as TNF and interleukin 1β (IL1β). In someembodiments, the GM-CSF inhibitor is an antibody that binds to andneutralizes circulating GM-CSF. In some embodiments, the antibody isselected from Lenzilumab; namilumab (AMG203); GSK3196165/MOR103/Otilimab(GSK/MorphoSys), KB002 and KB003 (KaloBios), MT203 (Micromet andNycomed), and MORAb-022/gimsilumab (Morphotek). In some embodiments, theantibody is a biosimilar of the same. In some embodiments, theantagonist is E21R, a modified form of GM-CSF that antagonizes thefunction of GM-CSF. In some embodiments, the inhibitor/antagonist is asmall molecule. In one embodiment, the CSF family member is M-CSF (alsoknown as macrophage colony-stimulating factor or CSF1). Non-limitingexamples of agents that inhibit or antagonize CSF1 include smallmolecules, antibodies, chimeric antigen receptors, fusion proteins, andother agents. In one embodiment, the CSF1 inhibitor or antagonist is ananti-CSF1 antibody. In one embodiment, the anti-CSF1 antibody isselected from those made by Roche (e.g., RG7155), Pfizer (PD-0360324),Novartis (MCS110/lacnotuzumab), or a biosimilar version of any one ofthe same. In some embodiments, the inhibitor or antagonist inactivatesthe activity of either the GM-CSF-R-alpha (aka CSF2R) or CSF1Rreceptors. In some embodiments, the inhibitor is selected fromMavrilimumab (formerly CAM-3001), a fully human GM-CSF Receptor amonoclonal antibody currently being developed by MedImmune, Inc.;cabiralizumab (Five Prime Therapeutics); LY3022855 (IMC-CS4)(Eli Lilly),Emactuzumab, also known as RG7155 or R05509554; FPA008, a humanized mAb(Five Prime/BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110(Novartis); PLX3397 (Plexxikon); ELB041/AFS98/TG3003 (ElsaLys Bio,Transgene), SNDX-6352 (Syndax). In some embodiments, the inhibitor orantagonist is expressed in CAR-T cells. In some embodiments, theinhibitor is a small molecule (e.g. heteroaryl amides, quinolinoneseries, pyrido-pyrimide series); BLZ945 (Novartis), PLX7486, ARRY-382,Pexidrtinib (also known as PLX3397) or5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine;GW 2580 (CAS 870483-87-7), KI20227 (CAS 623142-96-1), AC708 by AmbitSiosciences, or any CSF1R inhibitor listed in Cannarile et al. Journalfor ImmunoTherapy of Cancer 2017, 5:53 and US20180371093, incorporatedherein by reference for the inhibitors they disclose. Additionalneutralizing antibodies to GM-CSF or its receptor have been described inthe art, including in, for example, “GM-CSF as a target ininflammatory/autoimmune disease: current evidence and future therapeuticpotential” Hamilton, J. A. Expert Rev. Clin. Immunol., 2015; and“Targeting GM-CSF in inflammatory diseases” Wicks, I. P., Roberts, A. W.Nat. Rev. Rheumatol., 2016. In other embodiments, the agent is ananti-IL6 or anti-IL-6 receptor blocking agent, including tocilizumab andsiltuximab.

In one aspect, the therapeutic agent is a chemotherapeutic agent.Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL™, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®,Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate;CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO);retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™,(alitretinoin); ONTAK™ (denileukin diftitox); esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. In some aspects, compositionscomprising CAR- and/or TCR-expressing immune effector cells disclosedherein may be administered in conjunction with an anti-hormonal agentthat acts to regulate or inhibit hormone action on tumors such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above. Combinations of chemotherapeutic agents are also administeredwhere appropriate, including, but not limited to CHOP, i.e.,Cyclophosphamide (Cytoxan®), Doxorubicin (hydroxydoxorubicin),Vincristine (Oncovin®), and Prednisone.

The (chemo) therapeutic agent may be administered at the same time orwithin one week after the administration of the engineered cell ornucleic acid. In other aspects, the (chemo) therapeutic agent isadministered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1week to 12 months after the administration of the engineered cell ornucleic acid. In some aspects, the (chemo) therapeutic agent isadministered at least 1 month before administering the cell or nucleicacid. In some aspects, the methods further comprise administering two ormore chemotherapeutic agents.

A variety of additional therapeutic agents may be used inconjunction/combination with the compositions or agents/treatmentsdescribed herein. For example, potentially useful additional therapeuticagents include PD-1 inhibitors such as nivolumab (OPDIVO®),pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), andatezolizumab (Roche), tocilizumab (with and without corticosteroids),inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R GM-CSF, CSF1, GM-CSFR, orCSF1R (anti-CSF1 antibody is selected from those made by Roche (e.g.,RG7155), Pfizer (PD-0360324), Novartis (MCS110/lacnotuzumab),Mavrilimumab (formerly CAM-3001), a fully human GM-CSF Receptor amonoclonal antibody currently being developed by MedImmune, Inc.;cabiralizumab (Five Prime Therapeutics); LY3022855 (IMC-CS4)(Eli Lilly),Emactuzumab, also known as RG7155 or R05509554; FPA008, a humanized mAb(Five Prime/BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110(Novartis); PLX3397 (Plexxikon); ELB041/AFS98/TG3003 (ElsaLys Bio,Transgene), SNDX-6352 (Syndax). In some aspects, the inhibitor orantagonist is expressed in CAR-T cells. In some aspects, the inhibitoris a small molecule (e.g. heteroaryl amides, quinolinone series,pyrido-pyrimide series); BLZ945 (Novartis), PLX7486, ARRY-382,Pexidrtinib (also known as PLX3397) or5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine;GW 2580 (CAS 870483-87-7), KI20227 (CAS 623142-96-1), AC708 by AmbitSiosciences, or any CSF1R inhibitor listed in Cannarile et al. Journalfor ImmunoTherapy of Cancer 2017, 5:53 and US20180371093, incorporatedherein by reference for the inhibitors they disclose. Additionalneutralizing antibodies to GM-CSF or its receptor have been described inthe art,). Additional therapeutic agents suitable for use in combinationwith the compositions or agents/treatments and methods disclosed hereininclude, but are not limited to, ibrutinib (IMBRUVICA®), ofatumumab(ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab(HERCEPTIN®), trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®),cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab, ibritumomab,ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab,erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib,lenalidomide, axitinib, masitinib, pazopanib, sunitinib, sorafenib,tocilizumab, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib,nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib,tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib,dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib,ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib,binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide,denileukin diftitox, mTOR inhibitors such as Everolimus andTemsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDKinhibitors such as CDK inhibitor (palbociclib).

The composition or agents/treatments comprising immune cells are, or maybe, administered with an anti-inflammatory agent. Anti-inflammatoryagents or drugs may include, but are not limited to, steroids andglucocorticoids (including betamethasone, budesonide, dexamethasone,hydrocortisone acetate, corticosteroid, hydrocortisone, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone),nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin,ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNFmedications, cyclophosphamide and mycophenolate. Exemplary NSAIDsinclude ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, andsialylates. Exemplary analgesics include acetaminophen, oxycodone,tramadol of proporxyphene hydrochloride. Exemplary glucocorticoidsinclude cortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisolone, or prednisone. Exemplary biological response modifiersinclude molecules directed against cell surface markers (e.g., CD4, CD5,etc.), cytokine inhibitors, such as the TNF antagonists, (e.g.,etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®),chemokine inhibitors and adhesion molecule inhibitors. The biologicalresponse modifiers include monoclonal antibodies as well as recombinantforms of molecules. Exemplary DMARDs include azathioprine,cyclophosphamide, cyclosporine, methotrexate, penicillamine,leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin)and intramuscular), and minocycline.

The compositions or agents/treatments described herein may beadministered in conjunction with a cytokine and/or a cytokine modulatoras an additional therapeutic agent. Examples of cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor (HGF); fibroblast growth factor (FGF); prolactin;placental lactogen; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors(NGFs) such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO, Epogen®, Procrit®);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture, and biologically active equivalents of the native sequencecytokines. In one embodiment, the compositions described herein areadministered in conjunction with a steroid or corticosteroid.

Corticosteroid treatment may be used for treatment of adverse events.Corticosteroids (or any other steroids, as well as any other treatmentfor adverse events) may be used prophylactically, before any symptoms ofadverse events are detected and/or after detection of adverse events.They may be administered one or more days prior to T celladministration, on the day of T cell administration (before, after,and/or during T cell administration), and/or after T celladministration. They may be administered prior to, during, or afterconditioning therapy. Any corticosteroid may be appropriate for thisuse. In one embodiment, the corticosteroid is dexamethasone. In someembodiments, the corticosteroid is methylprednisolone. In someembodiments, the two are administered in combination. In someembodiments, glucocorticoids include synthetic and non-syntheticglucocorticoids. Exemplary glucocorticoids include, but are not limitedto: alclomethasones, algestones, beclomethasones (e.g. beclomethasonedipropionate), betamethasones (e.g. betamethasone 17 valerate,betamethasone sodium acetate, betamethasone sodium phosphate,betamethasone valerate), budesonides, clobetasols (e.g. clobetasolpropionate), clobetasones, clocortolones (e.g. clocortolone pivalate),cloprednols, corticosterones, cortisones and hydrocortisones (e.g.hydrocortisone acetate), cortivazols, deflazacorts, desonides,desoximethasones, dexamethasones (e.g. dexamethasone 21-phosphate,dexamethasone acetate, dexamethasone sodium phosphate), diflorasones(e.g. diflorasone diacetate), diflucortolones, difluprednates,enoxolones, fluazacorts, flucloronides, fludrocortisones (e.g.,fludrocortisone acetate), flumethasones (e.g. flumethasone pivalate),flunisolides, fluocinolones (e.g. fluocinolone acetonide),fluocinonides, fluocortins, fluocortolones, fluorometholones (e.g.fluorometholone acetate), fluperolones (e.g., fluperolone acetate),fluprednidenes, flupredni solones, flurandrenolides, fluticasones (e.g.fluticasone propionate), formocortals, halcinonides, halobetasols,halometasones, halopredones, hydrocortamates, hydrocortisones (e.g.hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisoneacetate, hydrocortisone buteprate, hydrocortisone butyrate,hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisoneprobutate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, hydrocortisone valerate), loteprednol etabonate,mazipredones, medrysones, meprednisones, methylpredni solones(methylprednisolone aceponate, methylprednisolone acetate,methylprednisolone hemisuccinate, methylprednisolone sodium succinate),mometasones (e.g., mometasone furoate), paramethasones (e.g.,paramethasone acetate), prednicarbates, prednisolones (e.g. prednisolone25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone21-hemisuccinate, prednisolone acetate; prednisolone farnesylate,prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide),prednisolone metasulphobenzoate, prednisolone steaglate, prednisolonetebutate, prednisolone tetrahydrophthalate), prednisones, prednivals,prednylidenes, rimexolones, tixocortols, triamcinolones (e.g.triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide, triamcinolone acetonide 21 palmitate, triamcinolonediacetate). These glucocorticoids and the salts thereof are discussed indetail, for example, in Remington's Pharmaceutical Sciences, A. Osol,ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980) and Remington: TheScience and Practice of Pharmacy, 22nd Edition, Lippincott Williams &Wilkins, Philadelphia, Pa. (2013) and any other editions, which arehereby incorporated by reference. In some embodiments, theglucocorticoid is selected from among cortisones, dexamethasones,hydrocortisones, methylprednisolones, prednisolones and prednisones. Inan embodiment, the glucocorticoid is dexamethasone. In otherembodiments, the steroid is a mineralcorticoid. Any other steroid may beused in the methods provided herein.

The one or more corticosteroids may be administered at any dose andfrequency of administration, which may be adjusted to the severity/gradeof the adverse event (e.g., CRS and NE). Tables 13, 14 and 16 provideexamples of dosage regimens for management of CRS and NE. In anotherembodiment, corticosteroid administration comprises oral or IVdexamethasone 10 mg, 1-4 times per day. Another embodiment, sometimesreferred to as “high-dose” corticosteroids, comprises administration ofIV methylprednisone 1 g per day alone, or in combination withdexamethasone. In some embodiments, the one or more cortico steroids areadministered at doses of 1-2 mg/kg per day.

The corticosteroid may be administered in any amount that is effectiveto ameliorate one or more symptoms associated with the adverse events,such as with the CRS or neurotoxicity. The corticosteroid, e.g.,glucocorticoid, can be administered, for example, at an amount betweenat or about 0.1 and 100 mg, per dose, 0.1 to 80 mg, 0.1 to 60 mg, 0.1 to40 mg, 0.1 to 30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5mg, 0.2 to 40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10mg, 0.2 to 5 mg, 0.4 to 40 mg, 0.4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg,0.4 to 10 mg, 0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg or 1 to10 mg, to a 70 kg adult human subject. Typically, the corticosteroid,such as a glucocorticoid is administered at an amount between at orabout 0.4 and 20 mg, for example, at or about 0.4 mg, 0.5 mg, 0.6 mg,0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg,18 mg, 19 mg or 20 mg per dose, to an average adult human subject.

In some embodiments, the corticosteroid may be administered, forexample, at a dosage of at or about 0.001 mg/kg (of the subject), 0.002mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg,0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg,0.055 mg/kg, 0.06 mg/kg, 0.065 mg/kg, 0.07 mg/kg, 0.075 mg/kg, 0.08mg/kg, 0.085 mg/kg, 0.09 mg/kg, 0.095 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1 mg/kg, 1.05 mg/kg, 1.1mg/kg, 1.15 mg/kg, 1.20 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg or 1.4mg/kg, to an average adult human subject, typically weighing about 70 kgto 75 kg.

Generally, the dose of corticosteroid administered is dependent upon thespecific corticosteroid, as a difference in potency exists betweendifferent corticosteroids. It is typically understood that drugs vary inpotency, and that doses can therefore vary, in order to obtainequivalent effects. Equivalence in terms of potency for variousglucocorticoids and routes of administration. is well known. Informationrelating to equivalent steroid dosing (in a non-chronotherapeuticmanner) may be found in the British National Formulary (BNF) 37, March1999.

In some embodiments, the adverse events/reactions may be chosen from oneor more of the following:

Adverse Event/Reaction Immune System Disorders Blood and LymphaticSystem Disorders Cytokine release syndrome Coagulopathy^(a)Hypogammaglobulinemia^(k) Cardiac Disorders Infections and InfestationsTachycardias^(b) Infection - pathogen unspecified Bradycardias^(c) Viralinfections Non-ventricular Arrhythmias^(d) Bacterial infectionsGastrointestinal Disorders Metabolism and nutrition disorders NauseaDecreased appetite Constipation Musculoskeletal pain¹ Diarrhea Motordysfunction^(m) Abdominal pain^(e) Psychiatric Disorders Oral pain^(f)Nervous System Disorders Vomiting^(g) Encephalopathy^(n) DysphagiaTremor Pyrexia Headache^(o) Fatigue^(h) Aphasia^(p) Chills Dizziness^(q)Edema¹ Neuropathy^(r) Dry mouth Insomnia Pain^(J) Delirium^(s) ImmuneSystem Disorders Anxiety Cytokine release syndrome Renal and UrinaryDisorders Hypogammaglobulinemia^(k) Renal insufficiency^(t) Infectionsand Infestations Urine output decreased^(u) Infection - pathogenunspecified Hypoxia Viral infections Cough^(v) Bacterial infectionsDyspnea^(w) Metabolism and nutrition disorders Pleural effusionDecreased appetite Skin and Subcutaneous Tissue DisordersMusculoskeletal pain¹ Rash^(x) Motor dysfunction^(m) Vascular DisordersPsychiatric Disorders Hypotension^(y) Nervous System DisordersHypertension Encephalopathy^(n) Thrombosis^(z) Tremor HemorrageHeadache^(o) Aphasia^(p) Dizziness^(q) Neuropathy^(r) InsomniaDelirium^(s) Anxiety

Other adverse reactions include; Gastrointestinal disorders: dry mouth;Infections and infestations disorders: fungal infection; Metabolism andnutrition disorders: dehydration; Nervous system disorders: ataxia,seizure, increased intracranial pressure; Respiratory, thoracic andmediastinal disorders: respiratory failure, pulmonary edema; Skin andsubcutaneous tissue disorders: rash; Vascular disorders: hemorrhage.

In one embodiment, cytokine release syndrome symptoms include but arenot limited to, fever, rigors, fatigue, anorexia, myalgias, arthalgias,nausea, vomiting, headache, rash, diarrhoea, tachypnea, hypoxemia,tachycardia, hypotension, widened pulse pressure, early increasedcardiac output, late diminished cardiac output, hallucinations, tremor,altered gait, seizures and death. In one embodiment, a method forgrading CRS is described in Neelapu et al., Nat Rev Clin Oncol.15(1):47-62 (2018) and Lee, et al., Blood 2014; 124:188-195. In oneembodiment, Neurotoxicity/Neurologic events may be graded by the methoddescribed in Lee, et al, Blood 2014; 124: 188-195.

In some embodiments, the adverse events are managed with tocilizumab (oranother anti-IL6/IL6R agent/antagonist), a corticosteroid therapy, or ananti-seizure medicine for toxicity prophylaxis. In some embodiments, theadverse events are managed by one or more agent(s) selected frominhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, anti-thymocyte globulin,lenzilumab, mavrilimumab, cytokines, and anti-inflammatory agents.

In some embodiments, the present disclosure provides methods ofpreventing the development or reducing the severity of adverse reactionsto the T cell treatments of the disclosure. In some embodiments, thecell therapy is administered in with one or more agents that prevents,delays the onset of, reduces the symptoms of, treats the adverse events,which include cytokine release syndromes and neurologic toxicity. In oneembodiment, the agent has been described above. In other embodiments,the agent is described below. In some embodiments, the agent isadministered by one of the methods and doses described elsewhere in thespecification, before, after, or concurrently with the administration ofthe cells. In one embodiment, the agent(s) are administered to a subjectthat may be predisposed to the disease but has not yet been diagnosedwith the disease.

In this respect, the disclosed method may comprise administering a“prophylactically effective amount” of tocilizumab, of a corticosteroidtherapy, and/or of an anti-seizure medicine for toxicity prophylaxis. Insome embodiments, the method comprises administering inhibitors ofGM-CSF, CSF1, GM-CSFR, or CSF1R, lenzilumab, mavrilimumab, cytokines,and/or anti-inflammatory agents. The pharmacologic and/or physiologiceffect may be prophylactic, i.e., the effect completely or partiallyprevents a disease or symptom thereof. A “prophylactically effectiveamount” may refer to an amount effective, at dosages and for periods oftime necessary, to achieve a desired prophylactic result (e.g.,prevention of onset of adverse reactions).

In some embodiments, the method comprises management of adversereactions in any subject. In some embodiments, the adverse reaction isselected from the group consisting of cytokine release syndrome (CRS), aneurologic toxicity, a hypersensitivity reaction, a serious infection, acytopenia and hypogammaglobulinemia. In some embodiments, the signs andsymptoms of adverse reactions are selected from the group consisting offever, hypotension, tachycardia, hypoxia, and chills, include cardiacarrhythmias (including atrial fibrillation and ventricular tachycardia),cardiac arrest, cardiac failure, renal insufficiency, capillary leaksyndrome, hypotension, hypoxia, organ toxicity, hemophagocyticlymphohistiocytosis/macrophage activation syndrome (HLH/MAS), seizure,encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomniaanxiety, anaphylaxis, febrile neutropenia, thrombocytopenia,neutropenia, and anemia. In some embodiments, the patient has beenidentified and selected based on one or more of the biomarkers ofadverse events. In some embodiments, the patient has been identified andselected simply by the clinical presentation (e.g., presence and gradeof toxicity symptom). In some embodiments, the adverse events aremanaged by any one of the protocols of Tables 13, 14, 16, and 17.

In some embodiments, the method comprises preventing or reducing theseverity of CRS in a chimeric receptor treatment. In some embodiments,the engineered CAR T cells are deactivated after administration to thepatient. In some embodiments, the method comprises identifying CRS basedon clinical presentation. In some embodiments, the method comprisesevaluating for and treating other causes of fever, hypoxia, andhypotension. Patients who experience ≥Grade 2 CRS (e.g., hypotension,not responsive to fluids, or hypoxia requiring supplemental oxygenation)should be monitored with continuous cardiac telemetry and pulseoximetry. In some embodiments, for patients experiencing severe CRS,consider performing an echocardiogram to assess cardiac function. Forsevere or life-threatening CRS, intensive care supportive therapy may beconsidered. In some embodiments, the method comprises monitoringpatients at least daily for 7 days at the certified healthcare facilityfollowing infusion for signs and symptoms of CRS. In some embodiments,the method comprises monitoring patients for signs or symptoms of CRSfor 4 weeks after infusion. In some embodiments, the method comprisescounseling patients to seek immediate medical attention should signs orsymptoms of CRS occur at any time. In some embodiments, the methodcomprises instituting treatment with supportive care, tocilizumab ortocilizumab and corticosteroids as indicated at the first sign of CRS.

In some embodiments, the method comprises monitoring patients for signsand symptoms of neurologic toxicities. In some embodiments, the methodcomprises ruling out other causes of neurologic symptoms. Patients whoexperience ≥Grade 2 neurologic toxicities should be monitored withcontinuous cardiac telemetry and pulse oximetry. Provide intensive caresupportive therapy for severe or life-threatening neurologic toxicities.In some embodiments, the symptom of neurologic toxicity is selected fromencephalopathy, headache, tremor, dizziness, aphasia, delirium,insomnia, and anxiety.

In some embodiments, the cell treatment is administered before,during/concurrently, and/or after the administration of one or moreagents (e.g., steroids) or treatments (e.g., debulking) that treat andor prevent (are prophylactic) one or more symptoms of adverse events. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. In one embodiment, a prophylactically effectiveamount is used in subjects prior to or at an earlier stage of disease.In one embodiment, the prophylactically effective amount will be lessthan the therapeutically effective amount. In one embodiment, theadverse event treatment or prophylaxis is administered to any patientthat will receive, is receiving, or has received cell therapy. In someembodiments, the method of managing adverse events comprises monitoringpatients at least daily for 7 days at the certified healthcare facilityfollowing infusion for signs and symptoms of neurologic toxicities. Insome embodiments, the method comprises monitoring patients for signs orsymptoms of neurologic toxicities and/or CRS for 4 weeks after infusion.

In some embodiments, the disclosure provides two methods of managingadverse events in subjects receiving CAR T cell treatment with steroidsand anti-IL6/anti-IL-6R antibody/ies. In one embodiment, the disclosureprovides a method of adverse event management whereby corticosteroidtherapy is initiated for management of all cases of grade 1 CRS if therewas no improvement after 3 days and for all grade ≥1 neurologic events.In one embodiment, tocilizumab is initiated for all cases of grade 1 CRSif there is no improvement after 3 days and for all grade ≥2 neurologicevents. In one embodiment, the disclosure provides a method of reducingoverall steroid exposure in patients receiving adverse event managementafter CAR T cell administration, the method comprising initiation ofcorticosteroid therapy for management of all cases of grade 1 CRS ifthere was no improvement after 3 days and for all grade ≥1 neurologicevents and/or initiation of tocilizumab for all cases of grade 1 CRS ifthere is no improvement after 3 days and for all grade ≥2 neurologicevents. In one embodiment, the corticosteroid and tocilizumab areadministering in a regimen selected from those exemplified the Examplessection. In one embodiment, the disclosure provides that earlier steroiduse is not associated with increased risk for severe infection,decreased CAR T-cell expansion, or decreased tumor response.

In one embodiment, the disclosure supports the safety of levetiracetamprophylaxis in CAR T cell cancer treatment. In one embodiment, thecancer is NHL. In one embodiment, the cancer is R/R LBCL and thepatients receive KTE-X19. Accordingly, in one embodiment, the disclosureprovides a method of managing adverse events in patients treated withCAR T cells comprising administering to the patient a prophylacticdosage of an anti-seizure medication. In some embodiments, the patientsreceive levetiracetam (for example, 750 mg orally or intravenous twicedaily) starting on day 0 of the CAR T cell treatment (afterconditioning) and also at the onset of grade ≥2 neurologic toxicities,if neurologic events occur after the discontinuation of prophylacticlevetiracetam. In one embodiment, if a patient does not experience anygrade ≥2 neurologic toxicities, levetiracetam is tapered anddiscontinued as clinically indicated. In one embodiment, levetiracetamprophylaxis is combined with any other adverse event managementprotocol.

In one embodiment, patients may receive levetiracetam (750 mg oral orintravenous twice daily) starting on day 0. At the onset of grade ≥2neurologic events, levetiracetam dose is increased to 1000 mg twicedaily. If a patient did not experience any grade ≥2 neurologic event,levetiracetam is tapered and discontinued as clinically indicated.Patients also receive tocilizumab (8 mg/kg IV over 1 hour [not to exceed800 mg]) on day 2. Further tocilizumab (±corticosteroids) may berecommended at the onset of grade 2 CRS in patients with comorbiditiesor older age, or otherwise in case of grade ≥3 CRS. For patientsexperiencing grade ≥2 neurologic events, tocilizumab is initiated, andcorticosteroids are added for patients with comorbidities or older age,or if there is any occurrence of a grade ≥3 neurologic event withworsening symptoms despite tocilizumab use.

In one embodiment, the disclosure provides that prophylactic steroid useappears to reduce the rate of severe CRS and NEs to a similar extent asearly steroid use administration. Accordingly, the disclosure provides amethod for adverse event management in CAR T-cell therapy whereinpatients receive dexamethasone 10 mg PO on Days 0 (prior to infusion),1, and 2. Steroids may also administered starting at Grade 1 NE, and forGrade 1 CRS when no improvement is observed after 3 days of supportivecare. Tocilizumab may also administered for Grade ≥1 CRS if noimprovement is observed after 24 hours of supportive care. In oneembodiment, the disclosure provides that adverse event management of CART-cell therapy with an antibody that neutralizes and/or depletes GM-CSFprevents or reduces treatment-related CRS and/or NEs in treatedpatients. In one embodiment, the antibody is lenzilumab.

In some embodiments, the adverse events are managed by theadministration of an agent/agents that is/are an antagonist or inhibitorof IL-6 or the IL-6 receptor (IL-6R). In some embodiments, the agent isan antibody that neutralizes IL-6 activity, such as an antibody orantigen-binding fragment that binds to IL-6 or IL-6R. For example, insome embodiments, the agent is or comprises tocilizumab (atlizumab) orsarilumab, anti-IL-6R antibodies. In some embodiments, the agent is ananti-IL-6R antibody described in U.S. Pat. No. 8,562,991. In some cases,the agent that targets IL-6 is an anti-TL-6 antibody, such assiltuximab, elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136),CPSI-2634, ARGX 109, FE301, FM101, or olokizumab (CDP6038), andcombinations thereof. In some embodiments, the agent may neutralize IL-6activity by inhibiting the ligand-receptor interactions. In someembodiments, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutein,such as one described in U.S. Pat. No. 5,591,827. In some embodiments,the agent that is an antagonist or inhibitor of IL-6/IL-6R is a smallmolecule, a protein or peptide, or a nucleic acid.

In some embodiments, other agents that may be used to manage adversereactions and their symptoms include an antagonist or inhibitor of acytokine receptor or cytokine. In some embodiments, the cytokine orreceptor is IL-10, TL-6, TL-6 receptor, IFNy, IFNGR, IL-2, IL-2R/CD25,MCP-1, CCR2, CCR4, MIP13, CCR5, TNFalpha, TNFR1, such as TL-6 receptor(IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 orCCR4), a TGF-beta receptor (TGF-beta I, II, or III), IFN-gamma receptor(IFNGR), MIP1P receptor (e.g., CCR5), TNF alpha receptor (e.g., TNFR1),IL-1 receptor (IL1-Ra/IL-1RP), or IL-10 receptor (IL-10R), IL-1, andIL-1Ralpha/IL-1beta. In some embodiments, the agent comprises situximab,sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429,sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In someembodiments, the agent, is an antagonist or inhibitor of a cytokine,such as transforming growth factor beta (TGF-beta), interleukin 6(TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNF alpha, IL-1,interferon gamma (IFN-gamma), or monocyte chemoattractant protein-I(MCP-1). In some embodiments, the is one that targets (e.g. inhibits oris an antagonist of) a cytokine receptor, such as TL-6 receptor (IL-6R),IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), aTGF-beta receptor (TGF-beta I, II, or III), IFN-gamma receptor (IFNGR),MIP1P receptor (e.g., CCR5), TNF alpha receptor (e.g., TNFR1), IL-1receptor (IL1-Ra/IL-1RP), or IL-10 receptor (IL-10R) and combinationsthereof. In some embodiments, the agent is administered by one of themethods and doses described elsewhere in the specification, before,after, or concurrently with the administration of the cells.

In some embodiments, the agent is administered in a dosage amount offrom or from about 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg, 2 mg/kg to 6mg/kg, 2 mg/kg to 4 mg/kg or 6 mg/kg to 8 mg/kg, each inclusive, or theagent is administered in a dosage amount of at least or at least aboutor about 2 mg/kg, 4 mg/kg, 6 mg/kg or 8 mg/kg. In some embodiments, isadministered in a dosage amount from about 1 mg/kg to 12 mg/kg, such asat or about 10 mg/kg. In some embodiments, the agent is administered byintravenous infusion. In one embodiment, the agent is tocilizumab. Insome embodiments, the (agent(s), e.g., specifically tocilizumab) is/areadministered by one of the methods and doses described elsewhere in thespecification, before, after, or concurrently with the administration ofthe cells.

In some embodiments, the method comprises identifying CRS based onclinical presentation. In some embodiments, the method comprisesevaluating for and treating other causes of fever, hypoxia, andhypotension. If CRS is observed or suspected, it may be managedaccording to the recommendations in protocol A, which may also be usedin combination with the other treatments of this disclosure, includingNeutralization or Reduction of the CSF/CSFR1 Axis. Patients whoexperience ≥Grade 2 CRS (e.g., hypotension, not responsive to fluids, orhypoxia requiring supplemental oxygenation) should be monitored withcontinuous cardiac telemetry and pulse oximetry. In some embodiments,for patients experiencing severe CRS, consider performing anechocardiogram to assess cardiac function. For severe orlife-threatening CRS, intensive care supportive therapy may beconsidered. In some embodiments, a biosimilar or equivalent oftocilizumab may be used instead of tocilizumab in the methods disclosedherein. In other embodiments, another anti-IL6R may be used instead oftocilizumab.

In some embodiments, adverse events are managed according to thefollowing protocol (protocol A):

CRS Grade (a) Tocilizumab Corticosteroids Grade 1 N/A N/A Symptomsrequire symptomatic treatment only (e.g., fever, nausea, fatigue,headache, myalgia, malaise). Grade 2 Administer tocilizumab (c) Manageper Grade 3 if no Symptoms require and respond 8 mg/kg IV over 1 hour(not to improvement within 24 hours to moderate intervention. exceed 800mg). after starting tocilizumab. Oxygen requirement less than Repeattocilizumab every 40% FiO₂ or hypotension 8 hours as needed if notresponsive to fluids or low- responsive to IV fluids or dose of onevasopressor or increasing supplemental Grade 2 organ toxicity (b).oxygen. Limit to a maximum of 3 doses in a 24-hour period; maximum totalof 4 doses if no clinical improvement in the signs and symptoms of CRS.Grade 3 Per Grade 2 Administer methylprednisolone Symptoms require andrespond 1 mg/kg IV twice daily or to aggressive intervention. equivalentdexamethasone Oxygen requirement greater (e.g., 10 mg IV every 6 hours).than or equal to 40% FiO₂ or Continue corticosteroids use hypotensionrequiring high- until the event is Grade 1 or dose or multiplevasopressors less, then taper over 3 days. or Grade 3 organ toxicity orIf not improving, manage as Grade 4 transaminitis. Grade 4. Grade 4 PerGrade 2 Administer methylprednisolone Life-threatening symptoms. 1000 mgIV per day for 3 days; Requirements for ventilator if improves, thenmanage as support, continuous veno- above. venous hemodialysis Consideralternate (CWHD) or immunosuppressants if no Grade 4 organ toxicityimprovement or if condition (excluding transaminitis). worsens. (a) LeeDW et al., (2014). Current concepts in the diagnosis and management ofcytokine release syndrome. Blood. 2014 Jul. 10; 124(2): 188-195. (b)Refer to Table 2 for management of neurologic toxicity. (c) Refer toACEMTRA ® (tocilizumab) Prescribing Information for details,https://www.gene.com/download/pdf/actemra_prescribing.pdf (last accessedOct. 18, 2017). Initial U.S. approval is indicated to be in 2010.

Neurologic Toxicity

In some embodiments, the method comprises monitoring patients for signsand symptoms of neurologic toxicities. In some embodiments, the methodcomprises ruling out other causes of neurologic symptoms. Patients whoexperience ≥Grade 2 neurologic toxicities should be monitored withcontinuous cardiac telemetry and pulse oximetry. Provide intensive caresupportive therapy for severe or life-threatening neurologic toxicities.Consider non-sedating, anti-seizure medicines (e.g., levetiracetam) forseizure prophylaxis for any ≥Grade 2 neurologic toxicities. Thefollowing treatments may be used in combination with the othertreatments of this disclosure, including Neutralization or Reduction ofthe CSF/CSFR1 Axis.

In some embodiments, adverse events are managed according to thefollowing protocol (protocol B):

Grading Assessment Concurrent CRS No concurrent CRS Grade 2 Administertocilizumab per table above Administer dexamethasone 10 mg IV (protocolA) for management of Grade 2 every 6 hours. CRS. Continue dexamethasoneuse until the If no improvement within 24 hours after event is Grade 1or less, then taper over starting tocilizumab, administer 3 days.dexamethasone 10 mg IV every 6 hours if not already taking othersteroids. Continue dexamethasone use until the event is Grade 1 or less,then taper over 3 days. Consider non-sedating, anti-seizure medicines(e.g., levetiracetam) for seizure prophylaxis. Grade 3 Administertocilizumab per (protocol A) Administer dexamethasone 10 mg IV formanagement of Grade 2 CRS. every 6 hours. In addition, administerdexamethasone Continue dexamethasone use until the 10 mg IV with thefirst dose of tocilizumab event is Grade 1 or less, then taper over andrepeat dose every 6 hours. Continue 3 days. dexamethasone use until theevent is Grade 1 or less, then taper over 3 days. Consider non-sedating,anti-seizure medicines (e.g., levetiracetam) for seizure prophylaxis.Grade 4 Administer tocilizumab per (protocol A) Administermethylprednisolone 1000 mg for management of Grade 2 CRS. IV per day for3 days; if improves, then Administer methylprednisolone 1000 mg manageas above. IV per day with first dose of tocilizumab and continuemethylprednisolone 1000 mg IV per day for 2 more days; if improves, thenmanage as above. Consider non-sedating, anti-seizure medicines (e.g.,levetiracetam) for seizure prophylaxis.

Additional Safety Management Strategies with Corticosteroids

Administration of corticosteroids and/or tocilizumab at Grade 1 may beconsidered prophylactic. Supportive care may be provided in allprotocols at all CRS and NE severity grades. In one embodiment of aprotocol for management of adverse events related to CRS, tocilizumaband/or corticosteroids are administered as follows: Grade 1 CRS: notocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only in caseof comorbidities or older age); and/or corticosteroids (only in case ofcomorbidities or older age); Grade 3 CRS: tocilizumab; and/orcorticosteroids; Grade 4 CRS: tocilizumab; and/or corticosteroids. Inanother embodiment of a protocol for management of adverse eventsrelated to CRS, tocilizumab and/or corticosteroids are administered asfollows: Grade 1 CRS: tocilizumab (if no improvement after 3 days);and/or corticosteroids (if no improvement after 3 days); Grade 2 CRS:tocilizumab; and/or corticosteroids; Grade 3 CRS: tocilizumab; and/orcorticosteroids; Grade 4 CRS: tocilizumab; and/or corticosteroids, highdose.

In one embodiment of a protocol for management of adverse events relatedto NE, tocilizumab and/or corticosteroids are administered as follows:Grade 1 NE: no tocilizumab; no corticosteroids; Grade 2 NE: notocilizumab; no corticosteroids; Grade 3 NE: tocilizumab; and/orcorticosteroids (only if no improvement to tocilizumab, standard dose);Grade 4 NE: tocilizumab; and/or corticosteroids. In another embodimentof a protocol for management of adverse events related to NE,tocilizumab and/or corticosteroids are administered as follows: Grade 1NE: no tocilizumab; and/or corticosteroids; Grade 2 NE: tocilizumab;and/or corticosteroids; Grade 3 NE: tocilizumab; and/or corticosteroids,high dose; Grade 4 NE: tocilizumab; and/or corticosteroids, high dose.In one embodiment, corticosteroid treatment is initiated at CRS grade ≥2and tocilizumab is initiated at CRS grade ≥2. In one embodiment,corticosteroid treatment is initiated at CRS grade ≥1 and tocilizumab isinitiated at CRS grade ≥1. In one embodiment, corticosteroid treatmentis initiated at NE grade ≥3 and tocilizumab is initiated at CRS grade≥3. In one embodiment, corticosteroid treatment is initiated at CRSgrade ≥1 and tocilizumab is initiated at CRS grade ≥2. In someembodiments, prophylactic use of tocilizumab administered on Day 2 maydecrease the rates of Grade ≥3 CRS. The one or more corticosteroids maybe administered at any dose and frequency of administration, which maybe adjusted to the severity/grade of the adverse event (e.g., CRS andNE). Tables 1 and 2 provide examples of dosage regimens for managementof CRS and NE, respectively. In another embodiment, corticosteroidadministration comprises oral or IV dexamethasone 10 mg, 1— 4 times perday. Another embodiment, sometimes referred to as “high-dose”corticosteroids, comprises administration of IV methylprednisone 1 g perday alone, or in combination with dexamethasone. In some embodiments,the one or more corticosteroids are administered at doses of 1-2 mg/kgper day. Generally, the dose of corticosteroid administered is dependentupon the specific corticosteroid, as a difference in potency existsbetween different corticosteroids. It is typically understood that drugsvary in potency, and that doses may therefore vary, in order to obtainequivalent effects. Equivalence in terms of potency for variousglucocorticoids and routes of administration. is well known. Informationrelating to equivalent steroid dosing (in a non-chronotherapeuticmanner) may be found in the British National Formulary (BNF) 37, March1999. The application also provides dosages and administrations of cellsprepared by the methods of the application, for example, an infusion bagof CD19-directed genetically modified autologous T cell immunotherapy,comprises a suspension of chimeric antigen receptor (CAR)-positive Tcells in approximately 68 mL for infusion. In some embodiments, the CART cells are formulated in approximately 40 mL for infusion. In someembodiments, the CAR T cell product is formulated in a total volume of35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 500, 700, 800, 900, 1000 mL. In one aspect, the dosage andadministration of cells prepared by the methods of the application, forexample, an infusion bag of CD19-directed genetically modifiedautologous T cell immunotherapy, comprises a suspension of 1×10⁶ CAR-Tpositive cells in approximately 40 mL. The target dose may be betweenabout 1×10⁶ and about 2×10⁶ CAR-positive viable T cells per kg bodyweight, with a maximum of 2×10⁸ CAR-positive viable T cells.

In some embodiments, the dosage form comprises a cell suspension forinfusion in a single-use, patient-specific infusion bag; the route ofadministration is intravenous; the entire contents of each single-use,patient-specific bag is infused by gravity or a peristaltic pump over 30minutes. In one embodiment, the dosing regimen is a single infusionconsisting of 2.0×10⁶ anti-CD19 CAR T cells/kg of body weight (±20%),with a maximum dose of 2×10⁸ anti-CD19 CART cells (for subjects ≥100kg). In some embodiments, the T cells that make up the dose are CD19CAR-T cells.

In some embodiments, the CD19-directed T cell immunotherapy is KTE-X19,which is prepared as described elsewhere in this application. In oneembodiment, KTE-X19 may be used for treatment of MCL, ALL, CLL, SLL, andany other B cell malignancy. In some embodiment, the CD19-directedgenetically modified autologous T cell immunotherapy is Axi-cel™(YESCARTA®, axicabtagene ciloleucel) prepared by one of the methods ofthe application. Amounts of CAR T cells, dosage regimens, methods ofadministration, subjects, cancers, that fall within the scope of thesemethods are described elsewhere in this application, alone or incombination with another chemotherapeutic agent, with or withoutpreconditioning, and to any of the patients described elsewhere in theapplication

The following examples are intended to illustrate various aspects of theapplication. As such, the specific aspects discussed are not to beconstrued as limitations on the scope of the application. For example,although the Examples below are directed to T cells transduced with ananti-CD19 chimeric antigen receptor (CAR), one skilled in the art wouldunderstand that the methods described herein may apply to immune cellstransduced with any CAR. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of application, and it is understood that suchequivalent aspects are to be included herein. Further, all referencescited in the application are hereby incorporated by reference in theirentirety, as if fully set forth herein.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, dictionaries,documents, manuscripts, genomic database sequences, and scientificliterature cited herein are hereby incorporated by reference.

Other features and advantages of the disclosure will be apparent fromthe Drawings and the following Detailed Description, including theExamples.

EXAMPLES Example 1

In this study, patients with R/R MCL who received 1-5 prior therapies,including a Bruton Tyrosine Kinase Inhibitor (BTKi), were treated withautologous anti-CD19 CAR-T cells.

Eligible patients (aged ≥18 years) with R/R MCL had an ECOG score of 0-1and ≤5 prior therapies, including chemotherapy, an anti-CD20 antibody,and a BTK inhibitor (BTKi). Patients underwent leukapheresis andchemotherapy (cyclophosphamide 300 mg/m²/d and fludarabine 30 mg/m²/dfor 3 days) followed by an infusion of CD19 CAR-T at a target dose of2×10⁶ CAR T cells/kg. Patients may have received bridging therapy withdexamethasone, ibrutinib, or acalabrutinib after leukapheresis andbefore chemotherapy. The primary endpoint was objective response rate(ORR [complete response (CR)+partial response (PR)]) according to theLugano Classification. Interim efficacy endpoints wereinvestigator-assessed using the revised IWG Response Criteria forMalignant Lymphoma. Key secondary endpoints were duration of response(DOR), progression-free survival (PFS), OS, frequency of adverse events(AEs), levels of CAR T cells in blood, and levels of cytokines in serum.

28 patients received CD19 CAR-T cells with ≥1 year of follow-up (median13.2 months [range, 11.5-18.5]). Forty-three percent of patients hadECOG score of 1, 21% had blastoid morphology, 82% had stage IV disease,50% had intermediate/high-risk MIPI, 86% received a median of 4 (range,1-5) prior therapies, and 57% were refractory to last prior therapy. In20/28 patients, the median Ki-67 index was 38% (range, 5%-80%). Eightpatients received bridging therapy; all had disease presentpost-bridging. ORR was 86% (95% CI, 67%-96%) with a CR rate of 57% (95%CI, 37%-76%). 75% of responders remained in response and 64% of treatedpatients had ongoing responses. The 12-month estimates of DOR, PFS andOS were 83% (95% CI, 60%-93%), 71% (95% CI, 50%-84%), 86% (95% CI,66%-94%), respectively and the medians were not reached. Grade ≥3 AEs(≥20% of patients) were anemia (54%), platelet count decreased (39%),neutropenia (36%), neutrophil count decreased (32%), white blood cellcount decreased (29%), encephalopathy (25%), and hypertension (21%).Grade 3/4 cytokine release syndrome (CRS) assessed by Lee D W, et al.Blood 2014; 124:188 was reported in 18% of patients, manifesting ashypotension (14%), hypoxia (14%), and pyrexia (11%). Grade 3/4neurologic events (NE) were reported in 46% of patients and includedencephalopathy (25%), confusional state (14%), and aphasia (11%). NoGrade 5 CRS or NE occurred. All CRS events and most NE (15/17 patients)were reversible. Median time to onset and resolution of CRS was 2 days(range, 1-7) and 13 days (range, 4-60), respectively. Median time toonset of NE was 6 days (range, 1-15) and median time to resolution was20 days (range, 9-99). Median CAR T cell levels as measured by peak andarea under the curve were 99 cells/μL (range, 0.4-2589) and 1542cells/μL (range, 5.5-27239), respectively. Peak CAR T cell expansion wasobserved between Days 8 and 15 and declined over time.

Example 2

This example provided additional analysis to the studies describedabove. Eligible patients were aged ≥18 years with pathologicallyconfirmed MCL with documentation of either cyclin D1 overexpression orpresence of t(11;14), and were relapsed/refractory to 1-5 prior regimensfor MCL. Prior therapy must have included anthracycline orbendamustine-containing chemotherapy, an anti-CD20 monoclonal antibody,and ibrutinib or acalabrutinib. All patients received prior BTKi.Although patients must have had prior BTKi therapy, it was not requiredas the last line of therapy before study entry, and patients were notrequired to be refractory to BTKi therapy. Eligible patients had anabsolute lymphocyte count ≥100/μL Patients who underwent autologous SCTwithin 6 weeks of CD19 CAR-T infusion or had previous CD19-targetedtherapy or allogeneic SCT were excluded.

Additional inclusion criteria included: at least 1 measurable lesion.Lesions that had been previously irradiated were considered measurableonly if progression had been documented following completion ofradiation therapy; If the only measurable disease was lymph nodedisease, at least 1 lymph node should have been ≥2 cm; Magneticresonance imaging (MRI) of the brain showing no evidence of centralnervous system (CNS) lymphoma; At least 2 weeks or 5 half-lives,whichever is shorter, must have elapsed since any prior systemic therapyor BTKi (ibrutinib or acalabrutinib) at the time the patient was plannedfor leukapheresis, except for systemic inhibitory/stimulatory immunecheckpoint therapy; At least 3 half-lives must have elapsed from anyprior systemic inhibitory/stimulatory immune checkpoint molecule therapyat the time the patient was planned for leukapheresis (eg, ipilimumab,nivolumab, pembrolizumab, atezolizumab, OX40 agonists, 4-1BB agonists);Toxicities due to prior therapy must have been stable and recovered to≤Grade 1 (except for clinically non-relevant toxicities such asalopecia); Eastern Cooperative Oncology Group (ECOG) performance statusof 0 or 1; Absolute neutrophil count (ANC)≥1 000/uL; Platelet count ≥75000/uL; Absolute lymphocyte count ≥100/uL; Adequate renal, hepatic,pulmonary, and cardiac function defined as: Creatinine clearance (asestimated by Cockcroft Gault)≥60 cc/min; Serum alanineaminotransferase/aspartate aminotransferase ≤2.5 upper limit of normal(ULN); Total bilirubin ≤1.5 mg/dl, except in patients with Gilbert'ssyndrome; Cardiac ejection fraction ≥50%, no evidence of pericardialeffusion as determined by an echocardiogram (ECHO), and no clinicallyrelevant electrocardiogram (ECG) findings; No clinically relevantpleural effusion; Baseline oxygen saturation >92% on room air; andFemales of childbearing potential must have had a negative serum orurine pregnancy test. Females who had undergone surgical sterilizationor who had been postmenopausal for at least 2 years were not consideredto be of childbearing potential.

Additional exclusion criteria included: History of malignancy other thannonmelanomatous skin cancer or carcinoma in situ (eg, cervix, bladder,breast) unless disease-free for at least 3 years; History of allogeneicstem cell transplantation; Prior CAR therapy or other geneticallymodified T-cell therapy; History of severe, immediate hypersensitivityreaction attributed to aminoglycosides; Presence of fungal, bacterial,viral, or other infection that was uncontrolled or requiring intravenous(IV) antimicrobials for management. Simple urinary tract infection (UTI)and uncomplicated bacterial pharyngitis were permitted if responding toactive treatment and after consultation with the medical monitor;History of human immunodeficiency virus (HIV) infection or acute orchronic active hepatitis B or C infection. Patients with a history ofhepatitis infection must have had cleared their infection as determinedby standard serological and genetic testing; Presence of any in-dwellingline or drain (eg, percutaneous nephrostomy tube, in-dwelling Foleycatheter, biliary drain, or pleural/peritoneal/pericardial catheter).Ommaya reservoirs and dedicated central venous access catheters, such asa Port-a-Cath or Hickman catheter, were permitted; Patients withdetectable cerebrospinal fluid malignant cells or brain metastases orwith a history of CNS lymphoma, cerebrospinal fluid malignant cells, orbrain metastases; History or presence of CNS disorder, such as seizuredisorder, cerebrovascular ischemia/hemorrhage, dementia, cerebellardisease, cerebral edema, posterior reversible encephalopathy syndrome,or any autoimmune disease with CNS involvement; History of myocardialinfarction, cardiac angioplasty or stenting, unstable angina, activearrhythmias, or other clinically relevant cardiac disease within 12months of enrollment; Patients with cardiac atrial or cardiacventricular lymphoma involvement; History of symptomatic deep veinthrombosis or pulmonary embolism within the last 6 months of enrollment;Possible requirement for urgent therapy due to ongoing or impendingoncologic emergency (eg, tumor mass effect, tumor lysis syndrome);Primary immunodeficiency; Any medical condition likely to interfere withassessment of safety or efficacy of study treatment; History of severeimmediate hypersensitivity reaction to any of the agents used in thisstudy; Live vaccine ≤6 weeks prior to planned start of conditioningregimen; Women of childbearing potential who were pregnant orbreastfeeding because of the potentially dangerous effects of thepreparative chemotherapy on the fetus or infant; Patients of bothgenders who were not willing to practice birth control from the time ofconsent through 6 months after the completion of the CD19 CAR-T celltreatment; In the investigator's judgment, the patient was unlikely tocomplete all protocol-required study visits or procedures, includingfollow-up visits, or comply with the study requirements forparticipation; and History of autoimmune disease (eg Crohn's disease,rheumatoid arthritis, systemic lupus) resulting in end organ injury orrequiring systemic immunosuppression/systemic disease modifying agentswithin the last 2 years.

All patients underwent leukapheresis to obtain cells for CD19 CAR-T celltreatment manufacturing. The manufacturing process was modified relativeto that of axicabtagene ciloleucel to remove circulating lymphoma cellsthrough positive enrichment for CD4⁺/CD8⁺ cells. Conditioningchemotherapy with fludarabine (30 mg/m²/day) and cyclophosphamide (500mg/m²/day) was administered on days −5, −4, and −3 prior to a singleintravenous infusion of 2×10⁶ CAR T cells/kg of CD19 CAR-T cells on day0. The dose was informed from studies of axicabtagene ciloleucel inlarge B-cell lymphoma and CD19 CAR-T cells in acute lymphoblasticleukemia. Neelapu S S et al. The New England journal of medicine 2017;377:2531; Locke Fla. et al. Mol Ther 2017; 25:285; Shah B D et al.Journal of Clinical Oncology 2019; 37:(suppl; abstr 7006); and Lee D Wet al. Annals of oncology: official journal of the European Society forMedical Oncology/ESMO 2017; 28:1008PD, all of which are incorporatedherein by reference in their entirety. Following leukapheresis andbefore conditioning therapy, patients with high disease burden wereallowed to receive bridging therapy with dexamethasone or equivalentcorticosteroid, ibrutinib, or acalabrutinib at the investigator'sdiscretion, after which a repeat baseline positron emissiontomography-computed tomography (PET-CT) scan was performed. The goal ofbridging therapy was not to be curative but to keep patients stablethrough the manufacturing period. Hospitalization post-CD19 CAR-T cellinfusion was required through day 7.

The primary end point was the objective response rate (ORR [completeresponse (CR)+partial response] (PR)) as assessed by the IndependentRadiology Review Committee (IRRC) using the Lugano classification.Cheson et al., J Clin Oncol 2014; 32:3059-68. In order to confirm a CR,bone marrow evaluation in addition to PET-CT was required. Secondary endpoints included duration of response (DOR), progression-free survival(PFS), OS, investigator-assessed ORR according to Cheson, et al, J ClinOncol 2007; 25:579-86, incidence of adverse events (AEs), levels of CARTcells in blood and cytokines in serum, and change in scores over time inthe European Quality of Life-5 Dimensions with 5 levels per dimension(EQ-5D-5L). CAR T-cell presence, expansion, and persistence and serumcytokines, as well as their associations with clinical outcomes, wereassessed as previously reported. Kochenderfer J N et al. J Clin Oncol2017; 35:1803-13; Locke F L et al. Mol Ther 2017; 25:285-95, both ofwhich are incorporate herein by reference in their entirety.

Changes in EQ-5D-5L scores from baseline to month 6 were assessed.Cytokine release syndrome (CRS) was graded according to Lee et al. Blood2014; 124:188, incorporate herein by reference in its entirety. Severityof AEs, including neurologic events and symptoms of CRS, was gradedusing the National Cancer Institute Common Terminology Criteria forAdverse Events, version 4.03. Minimal residual disease (MRD; 10⁻⁵sensitivity) was an exploratory analysis assessed in cryopreservedperipheral blood mononuclear cells at baseline and months 1, 3, and 6,and was analyzed by next-generation sequencing using the clonoSEQ assay(Adaptive Biotechnologies, Seattle, Wash.

For all patients, positron emission tomography-computed tomography(PET-CT) scans of disease-specific sites were required at baseline, 4weeks post-infusion, and at regular intervals during the posttreatmentperiod. A bone marrow aspirate/biopsy was required to confirm a completeresponse in patients with bone marrow disease involvement at baselineand in patients with indeterminate bone marrow involvement at baseline,or if no baseline bone marrow biopsy was done or the results wereunavailable. Patients with symptoms of CNS malignancy had lumbarpuncture performed at screening for examination of cerebral spinal fluid(CSF). Lumbar puncture was also performed as applicable for patientswith new onset of Grade ≥2 neurologic toxicities after anti-CD19 CART-cell infusion. In addition, for patients who sign an optional portionof the consent form, lumbar puncture for CSF collection was performed atbaseline prior to anti-CD19 CAR T-cell infusion and after anti-CD19 CART-cell infusion (Day 5±3 days); samples were submitted to the centrallaboratory and analyzed for changes in cytokine levels.

The primary analysis for efficacy was conducted after 60 patients wereenrolled, treated, and evaluated for response 6 months after the week 4disease assessment, as required by the protocol. This analysis had apower of ≥96% to distinguish between an active therapy with a 50% trueresponse rate and a therapy with a response rate of ≤25% with aone-sided alpha level of 0.025. An exact binomial test was used toanalyze ORR. All efficacy endpoints, including time-to-event endpointsas assessed using Kaplan-Meier estimates, were analyzed in the 60efficacy-evaluable patients described above. Safety analyses wereconducted in all treated patients (n=68). Associations between outcomesand CAR T-cell and cytokine levels were measured using the Wilcoxonrank-sum test; P values were adjusted using Holm's procedure. Fullanalysis set (N=74): Consisted of all enrolled/leukapheresed patientsand was used for the summary of patient disposition. Safety analysis set(n=68): Defined as all patients treated with any dose of anti-CD19 CAR Tcells. This analysis set was used for the summary of demographics andbaseline characteristics and all analysis of safety. Inferentialanalysis (efficacy evaluable) set (n=60): Consisted of the first 60treated CD19 CAR-T cell patients. This analysis set was used for thehypothesis testing of the primary endpoint of objective response rate atthe time of the primary analysis, as well as all other efficacyanalyses. The hypothesis for the primary endpoint was that the ORR toCD19 CAR-T cells using central assessment would be greater than theprespecified historical control rate of 25% at the 1-sided significancelevel of 0.025 using an exact binomial test. This hypothesis was to betested in the inferential analysis set. The historical control rate forORR was determined a priori based on 2 retrospective studies that werepublished at the time of the study protocol development. In these 2studies, outcomes after salvage therapy were evaluated in patients withrelapsed/refractory MCL who had progressed following treatment with aBTKi (a required prior therapy for study eligibility). These studiesshowed that patients with relapsed/refractory MCL who had ≥3 prior linesbefore receiving the BTKi had ORRs to salvage therapy of approximately25%. Wang M et al. Lancet 2018; 391:659; Martin P et al. Blood 2016;127:1559, both of which are incorporated herein by reference in theirentirety.

Seventy-four patients were enrolled; CD19 CAR-T cells were manufacturedfor 71 and administered to 68. Primary efficacy analysis conducted after60 patients were treated exhibited an ORR of 93% (67% completeresponses). At a median follow-up of 12.3 months (range, 7.0-32.3), 57%of patients remained in remission and median duration of response wasnot reached. The estimated 12-month progression-free survival andoverall survival rates were 61% and 83%, respectively. Common grade ≥3adverse events were cytopenias (94%) and infections (32%). Grade ≥3cytokine release syndrome and neurologic events occurred in 15% and 31%of patients, respectively; none were fatal. Two grade 5 infectiousadverse events occurred.

CD19 CAR-T cells were manufactured for 71 patients (96%) andadministered to 68 (92%). The median time from leukapheresis to deliveryof CD19 CAR-T cells to the study site was 16 days (range, 11-128). Onepatient who had CD19 CAR-T cells manufactured was treated withbendamustine-rituximab due to rapid PD following leukapheresis, makingthe patient ineligible for the study. After later developing PD, thepatient's original product was shipped from the manufacturing facility127 days after the initial leukapheresis date, arriving at the treatingsite 1 day later. Three patients with manufacturing issues did notproceed to an additional apheresis due to AE (n=1; deep veinthrombosis), death due to progressive disease (PD; n=1), or consentwithdrawal (n=1). Two additional patients discontinued prior toconditioning chemotherapy due to death from PD. After receivingconditioning chemotherapy, 1 patient with ongoing atrial fibrillation,an exclusion criterion, was deemed ineligible for CD19 CAR-T cellsinfusion. The median follow-up for efficacy-evaluable patients was 12.3months (range, 7.0-32.3); 28 patients had ≥24 months of follow-up.

The median age was 65 years (range, 38-79) and 57 (84%) of patients weremale. (Table 1) 65% had an ECOG performance status score of 0 and 35%of 1. Patients had high-risk features at baseline, including stage IVdisease (85%), blastoid or pleomorphic morphology (31%), Ki-67proliferation index ≥30% (40/49 [82%]) (Wang M L et al. The LancetOncology 2016; 17:48), and TP53 mutation (6/36 [17%]). Eighty-onepercent of patients had received ≥3 prior lines of therapy (median, 3[range 1-5]).

TABLE 1 Baseline patient characteristics Characteristic N = 68 Age,median (range), y 65 (38-79) >65 years, n (%) 39 (57) Male, n (%) 57(84) ECOG performance status score, n (%) 0 44 (65) 1 24 (35) Stage IVdisease, n (%) 58 (85) Bone marrow involvement, n (%) 37 (54) Splenicinvolvement, n (%) 23 (34) Extranodal disease, n (%)* 38 (56) Bulkydisease (≥10 cm), n (%) 7 (10) Simplified MIPI, n (%)^(†) Low risk 28(41) Intermediate risk 29 (43) High risk 9 (13) Missing 2 (3) MCLmorphology, n (%) Classical 40 (59) Pleomorphic 17 (25) Blastoid 4 (6)Other/Unknown^(††) 11 (16) Ki-67 proliferation index, median (range),%^(§) 65 (1-95) ≥30%, n/n (%) 40/49 (82) ≥50%, n/n (%) 34/49 (69) TP53mutation, n (%) 6/36 (17%) CD19 status, n/n (%) Positive 47/51 (92)Negative 4/51 (8) Number of prior therapies, median (range) 3 (1-5) ≥3prior lines of therapy, n (%) 55 (81) Prior therapy,^(¶) n (%) Anti-CD2068 (100) BTKi 68 (100) Ibrutinib 58 (85) Acalabrutinib 16 (24) Both 6(9) Anthracycline or bendamustine 67 (99) Anthracycline 49 (72)Bendamustine 37 (54) Autologous SCT 29 (43) Bortezomib 24 (35)Lenalidomide 19 (28) Other investigational agent 11 (16) Venetoclax 6(9) Relapsed/refractory subgroup, n (%) Relapsed after autologous SCT 29(43) Refractory to last prior therapy 27 (40) Relapsed after last priortherapy 12 (18) Refractory to ibrutinib, n (%) 38 (56) Refractory toacalabrutinib, n (%) 8 (12) *Excludes bone marrow and splenicinvolvement. ^(†)At diagnosis. ^(††)One patient was reported by theinvestigator to have kappa light chain restricted MCL at diagnosis.Morphology was reported as unknown for 10 patients. ^(§)Ki-67 data wasavailable for 49 patients at diagnosis. ^(¶)Induction plusconsolidation/maintenance and/or all treatments occurring betweensequential complete responses was counted as 1 regimen. BTKi, Brutontyrosine kinase inhibitor; ECOG, Eastern Cooperative Oncology Group;MCL, mantle cell lymphoma; MIPI, Mantle Cell Lymphoma InternationalPrognostic Index; SCT, stem cell transplant.

All patients had progressed on a BTKi (ibrutinib n=58; acalabrutinibn=16; both n=6), and 43% had prior autologous SCT (Table 2). Median timefrom end of last BTKi therapy excluding bridging to CD19 CAR-T cellinfusion was 88 days (range, 25-1047). Forty percent of patients wererefractory to last therapy, including 3 ibrutinib-intolerant patientswith confirmed progression after last therapy. Twenty-five patients(37%) received bridging therapy with ibrutinib (n=14), acalabrutinib(n=5), dexamethasone (n=12), and/or methylprednisolone (n=2).Post-bridging scans showed that most patients had tumor burden higherthan the median at screening.

TABLE 2 Bridging Therapies Characteristic N = 68 Any bridging therapy, n(%) 25 (37) Ibrutinib 14 (21) Acalabrutinib 5 (7) Dexamethasone 12 (18)Methylprednisolone 2 (3) Both BTKi and steroids, n (%) 6 (9) Ibrutinib +steroid 4 (6) Acalabrutinib + steroid 2 (3) BTKi, Bruton tyrosine kinaseinhibitor.

The IRRC-assessed ORR among the protocol-specified 60 patients treatedwith CD19 CAR-T with a minimum follow-up of 7 months was 93% (95% CI,84-98), with a 67% CR rate and 27% PR rate. High concordance (95%) wasobserved between IRRC-assessed and investigator-assessed ORR (Table 3).

TABLE 3 Response in Efficacy-Evaluable Patients Based on InvestigatorAssessment According to Cheson BD et al. J Clin Oncol 2007;25:57 and inIntent-to-Treat Patients by IRRC Review per the Lugano Classification(2014). Investigator-Assessed IRRC-Assessed Efficacy EvaluableIntent-to-Treat n (%) N = 60 N = 74 Objective response rate  53 (88) 63(85) Complete response  42 (70) 44 (59) Partial response  11 (18) 19(26) Stable disease   5 (8) 3 (4) Disease Progression   2 (3) 2 (3) NotAssessed*   0 (0) 6 (8) Concordance with IRRC-assessed ORR, %^(†) 95 N/AKappa coefficient (95% CI) 0.7 (0.4-1.0) N/A Concordance withIRRC-assessed CR rate, %^(†) 90 N/A Kappa coefficient (95% CI) 0.8(0.6-0.9) N/A *No assessment at the time of analysis. ^(†)Concordance isthe percentage of subjects whose IRRC-assessed read matchesinvestigator-assessed read. CR, complete response; IRRC, IndependentRadiology Review Committee; N/A, not applicable; ORR, objective responserate.

The IRRC-assessed ORR for all enrolled patients (n=74) was 85% (95% CI,75-92), with a 59% CR rate. ORR was consistent across key subgroups,including age, relapsed/refractory subgroup, number of prior therapies,MCL morphology, disease stage, extranodal disease, bone marrowinvolvement, simplified MIPI, CD19 positive, tumor burden, serum lactatedehydrogenase levels, TP53 mutational status, Ki-67 index, use oftocilizumab or steroids for AE management, and use of bridging therapy.The median time to initial response was 1.0 month (range, 0.8-3.1), andthe median time to CR was 3.0 months (range, 0.9-9.3). Of the 42patients who initially achieved PR or SD, 24 patients (57%), including21 with initial response of PR and 3 with initial response of SD,subsequently converted to a CR after a median of 2.2 months (range,1.8-8.3) following the initial response; 18 of these 24 patients remainin remission. MRD analysis was conducted in 29/60 patients (48%); 24/29patients (83% [19 CR; 5 PR]) were MRD negative at week 4, and 15/19patients (79%) with available data remained MRD negative at month 6. MRDwas unable to be assessed in all patients due to the lack ofavailability of a formalin-fixed paraffin-embedded tumor biopsy samplefor calibration, which was required by the methodology and used toestablish dominant rearranged IgH (VDJ or DJ), IgK, or IgL receptor genesequences tracked over time in blood. Two patients who progressed afterresponding to CD19 CAR-T cells received a second infusion approximately1 year and 2.6 years after the initial infusion; analysis of thesepatients is ongoing.

The median DOR has not been reached after a median follow-up of 12.3months (Median (95% CI); Not reached (8.6, NE). The median progressionfree survival (95% CI) was not reached (9.2, NE). The median overallsurvival (95% CI) was also not reached (24.0, NE). Fifty-seven percentof all patients and 78% CR patients remain in remission. However, thefirst 28 patients treated had a median follow-up of 27.0 months (range;25.3-32.3), with 43% in continued remission without additional therapy.Ongoing response rates were consistent across key covariates, includingage, MCL morphology, relapsed/refractory subgroup, Ki-67 index, diseasestage, extranodal disease, bone marrow involvement, simplified MIPI,TP53 mutation, CD19 positive, bridging therapy, tumor burden, and use oftocilizumab or steroids. The 3 patients with CD19− tumors at baselineachieved CR and remain in ongoing responses as of the data cutoff. Themedian PFS and OS were not reached, with estimated 12-month rates of 61%(95% CI, 45-74) and 83% (95% CI, 71-91), respectively. Although limitedin sample size, subgroup analysis of PFS showed that the 6-month PFSrate was consistent among patients with blastoid or pleomorphicmorphology, TP53 mutation, or Ki-67 index ≥50%. At the time of thisanalysis, 76% of all patients remain alive. Of the patients who had aresponse, 14 had PD. One patient who had a PR underwent allogeneic SCT.

This study showed an ORR of 93% in the protocol-specified 60 patientswith relapsed/refractory MCL, all of whom had relapsed after or wererefractory to BTKi therapy. This ORR included a 67% CR rate, after asingle infusion. After a median follow-up of 12.3 months, the median DORhad not been reached; 57% of all patients and 78% of CR patientsremained in response. Twenty-eight (28) patients treated with CD19 CAR-Tcells have had a longer median follow-up of 27 months (range,25.3-32.3), and 43% continued to be in remission without additionaltherapy. Response rates, including ongoing responses, were generallysimilar among key subgroups including patients with high-risk features.Patients with Ki-67 ≥50%, as well as patients with blastoid/pleomorphicmorphology or TP53 mutation had high ORR and 6-month PFS rates similarto the overall population, suggesting that CD19 CAR-T cell treatmentbenefited patients with typically worse prognosis.

All patients who responded after CAR T-cell infusion achieved T-cellexpansion. This expansion was not observed in non-responding patients,suggesting that response may have been related to sufficient CAR T cellexpansion. Similar to prior studies, CAR T-cell levels correlated withORR in the first 28 days, suggesting that higher expansion led to betterand perhaps deeper responses as indicated by the >80-fold higherpeak/AUC CAR T-cell levels in MRD negative compared with positivepatients. Response rates were also similar regardless of whetherbridging therapy was administered, and most patients with post-bridgingscans (87%) had an increase in SPD compared with pre-bridging scans.

All treated patients experienced ≥1 AE of any grade, with grade ≥3 AEsin 99% (Table 2). The most common AEs of any grade were pyrexia (94%),neutropenia (87%), thrombocytopenia (74%), and anemia (68%). The mostcommon grade ≥3 AEs were neutropenia (85%), thrombocytopenia (51%),anemia (50%), and infections (32%). Twenty-six percent of patients hadgrade ≥3 cytopenias present >90 days post-CD19 CAR-T cells, includingneutropenia (16%), thrombocytopenia (16%), and anemia (12%). CRSoccurred in 91% of patients (Table 4). No patient died due to CRS. Mostcases were grade 1/2 (76%), with grade ≥3 CRS occurring in 15% ofpatients. The most common grade ≥3 symptoms of CRS were hypotension(22%), hypoxia (18%), and pyrexia (11%). For CRS management, 59% ofpatients received tocilizumab, 22% received steroids, and 16% requiredvasopressors. The median time after infusion to the onset of any gradeand grade ≥3 CRS was 2 days (range, 1-13) and 4 days (range, 1-9),respectively; all events resolved within a median of 11 days.

TABLE 4 Adverse events, cytokine release syndrome, and neurologic eventsN = 68 n (%)* Any Grade Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Anyadverse event 68 (100) 0 (0) 1 (1) 11 (16) 52 (76) 2 (3) Pyrexia 64 (94)14 (21) 41 (60) 9 (13) 0 0 Neutropenia 59 (87) 0 (0) 1 (1) 11 (16) 47(69) 0 (0) Thrombocytopenia 50 (74) 9 (13) 6 (9) 11 (16) 24 (35) 0 (0)Anemia 46 (68) 0 12 (18) 34 (50) 0 0 Hypotension 35 (51) 4 (6) 16 (24)13 (19) 2 (3) 0 Chills 28 (41) 17 (25) 11 (16) 0 0 0 Hypoxia 26 (38) 2(3) 10 (15) 8 (12) 6 (9) 0 Cough 25 (37) 14 (21) 11 (16) 0 0 0Hypophosphatemia 25 (37) 2 (3) 8 (12) 15 (22) 0 0 Fatigue 24 (35) 10(15) 13 (19) 1 (1) 0 0 Headache 24 (35) 15 (22) 8 (12) 1 (1) 0 0 Tremor24 (35) 19 (28) 5 (7) 0 0 0 Hypoalbuminemia 23 (34) 5 (7) 17 (25) 1 (1)0 0 Hyponatremia 22 (32) 15 (22) 0 7 (10) 0 0 Nausea 22 (32) 11 (16) 10(15) 1 (1) 0 0 Alanine 21 (31) 13 (19) 2 (3) 5 (7) 1 (1) 0aminotransferase increased Encephalopathy 21 (31) 5 (7) 3 (4) 7 (10) 6(9) 0 Hypokalemia 21 (31) 12 (18) 4 (6) 3 (4) 2 (3) 0 Tachycardia 21(31) 14 (21) 7 (10) 0 0 0 CRS^(†) 62 (91) 20 (29) 32 (47) 8 (12) 2 (3) 0Symptoms Pyrexia 62 (100) 15 (24) 40 (65) 7 (11) 0 0 Hypotension 35 (56)4 (6) 16 (26) 14 (23) 1 (2) 0 Hypoxia 23 (37) 1 (2) 10 (16) 8 (13) 4 (6)0 Chills 21 (34) 12 (19) 9 (15) 0 0 0 Tachycardia 16 (26) 11 (18) 5 (8)0 0 0 Headache 15 (24) 7 (11) 8 (13) 0 0 0 Alanine 10 (16) 5 (8) 1 (2) 3(5) 1 (2) 0 aminotransferase increased Aspartate 9 (15) 4 (6) 0 (0) 5(8) 0 0 aminotransferase increased Fatigue 9 (15) 6 (10) 2 (3) 1 (2) 0 0Nausea 9 (15) 5 (8) 4 (6) 0 0 0 Any neurologic event 43 (63) 13 (19) 9(13) 15 (22) 6 (9) 0 Tremor 24 (35) 19 (28) 5 (7) 0 0 0 Encephalopathy21 (31) 5 (7) 3 (4) 7 (10) 6 (9) 0 Confusional state 14 (21) 3 (4) 3 (4)8 (12) 0 0 Aphasia 10 (15) 3 (4) 4 (6) 3 (4) 0 0 *Included are adverseevents occurring in ≥30% of patients, and symptoms of CRS and neurologicevents occurring in ≥15% of patients. ^(†)Percentages in the CRS rowswere calculated out the 62 patients who experienced CRS.

Sixty-three percent of patients experienced NE (Table 4). No patientdied from NE. Grade 1/2 NE occurred in 32% of patients and grade ≥3 NEin 31%. Common grade ≥3 NE were encephalopathy (19%), confusional state(12%), and aphasia (4%). One patient developed grade 4 cerebral edemaand fully recovered with aggressive multimodality therapy includingventriculostomy. Tocilizumab and steroids were used to treat NE in 26%and 38% of patients, respectively. The median time to onset of any gradeand grade ≥3 NE was 7 days (range, 1-32) and 8 days (range, 5-24),respectively. The median duration of NE was 12 days with events fullyresolving in 37/43 patients (86%). As of this analysis, 4 patients hadongoing events, including grade 1 tremor (n=3), grade 2 concentrationimpairment (n=1), and grade 1 dysesthesia (n=1). Serious AEs occurred in68% of patients (Table 5).

TABLE 5 Serious Adverse Events Occurring in at Least 3 Patients Seriousadverse N = 68 event, n (%) Any Grade Grade 1 Grade 2 Grade 3 Grade 4Grade 5 Any 46 (68) 2 (3) 7 (10) 20 (29) 13 (19) 2 (3) Encephalopathy 15(22) 2 (3) 1 (1) 6 (9) 6 (9) 0 Pyrexia 15 (22) 7 (10) 5 (7) 3 (4) 0 0Hypotension 11 (16) 0 3 (4) 6 (9) 2 (3) 0 Hypoxia 8 (12) 0 0 4 (6) 4 (6)0 Acute kidney injury 5 (7) 0 0 1 (1) 4 (6) 0 Confusional state 5 (7) 00 5 (7) 0 0 Pneumonia 5 (7) 0 0 5 (7) 0 0 Anemia 4 (6) 0 0 4 (6) 0 0Respiratory failure 4 (6) 0 0 0 4 (6) 0 Sepsis 4 (6) 0 0 1 (1) 3 (4) 0Aphasia 3 (4) 0 0 3 (4) 0 0 Pleural effusion 3 (4) 0 1 (1) 1 (1) 1 (1) 0Tachycardia 3 (4) 0 3 (4) 0 0 0

Thirty-two percent of patients experienced grade ≥3 infections. The mostcommon was pneumonia (9%) (Table 6).

TABLE 6 Infections Occurring in at Least 2 Patients N = 68 Infection, n(%) Any Grade Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Any 38 (56) 1 (1)15 (22) 17 (25) 4 (6)  2 (2)* Upper respiratory tract 9 (13) 0 (0) 8(12) 1 (1) 0 (0) 0 (0) infection Pneumonia 7 (10) 0 (0) 1 (1) 6 (9) 0(0) 0 (0) Sinusitis 5 (7) 0 (0) 5 (7) 0 (0) 0 (0) 0 (0) Sepsis 4 (6) 0(0) 0 (0) 1 (1) 3 (4) 0 (0) Oral candidiasis 4 (6) 0 (0) 4 (6) 0 (0) 0(0) 0 (0) Herpes zoster 3 (4) 0 (0) 3 (4) 0 (0) 0 (0) 0 (0) Influenza 3(4) 0 (0) 3 (4) 0 (0) 0 (0) 0 (0) Staphylococcal bacteremia 3 (4) 0 (0)0 (0) 2 (3) 0 (0) 1 (1) Cytomegalovirus infection 2 (3) 0 (0) 2 (3) 0(0) 0 (0) 0 (0) Fungal skin infection 2 (3) 1 (1) 1 (1) 0 (0) 0 (0) 0(0) Cellulitis 2 (3) 0 (0) 2 (3) 0 (0) 0 (0) 0 (0) Bronchitis 2 (3) 0(0) 1 (1) 1 (1) 0 (0) 0 (0) Nasopharyngitis 2 (3) 2 (3) 0 (0) 0 (0) 0(0) 0 (0) Tooth infection 2 (3) 0 (0) 0 (0) 2 (3) 0 (0) 0 (0) *Onepatient died from staphylococcus bacteremia. One patient died fromorganizing pneumonia (developed acute kidney injury in the setting ofinfection and during autopsy was found to have a previously undiagnosedpulmonary embolism in addition to organizing pneumonia).

Two cases of grade 2 cytomegalovirus infection occurred (3%). Grade 3hypogammaglobulinemia and grade 3 tumor lysis syndrome occurred in 1patient each (1%). Twenty-two patients (32%) received intravenousimmunoglobulin therapy. No cases of replication-competent retrovirus,EBV-associated lymphoproliferation, hemophagocytic lymphohistiocytosis,or CD19 CAR-T cells-related secondary cancers were reported. EQ-5Dscores revealed decrements from baseline in patient-reportedhealth-related quality of life at week 4, yet improvements in mobility,self-care, usual activities, and overall health (EQ-5D visual analoguescale) were observed by month 3, with overall health returning tobaseline or better in most patients by month 6 (Table 7).

TABLE 7 EQ-5D Summary by Visit EQ-5D Screening Week 4 Month 3 Month 6Mobility, n/n (%) Patients reporting no problems 53/62 (85) 25/51 (49)37/54 (69) 30/40 (75) Patients with deterioration from N/A 21/51 (41)13/54 (24)  8/40 (20) screening Self-care, n/n (%) Patients reporting noproblems 59/62 (95) 35/52 (67) 45/54 (83) 37/40 (93) Patients withdeterioration from N/A 16/52 (31)  9/54(17)  3/40 (8) screening Usualactivity, n/n (%) Patients reporting no problems 53/65 (82) 22/51 (43)38/55 (69) 30/41 (73) Patients with deterioration from N/A 25/51 (49)13/55 (24)  8/41 (20) screening Pain/Discomfort, n/n (%) Patientsreporting no problems 43/65 (66) 34/54 (63) 33/55 (60) 28/42 (67)Patients with deterioration from N/A  9/54 (17) 13/55 (24)  5/42 (12)screening Anxiety/Depression, n/n (%) Patients reporting no problems49/65 (75) 36/54 (67) 38/55 (69) 26/42 (62) Patients with deteriorationfrom N/A 11/54 (20) 12/55 (22) 10/42 (24) screening EQ-5D VAS* n 65 5255 42 Mean (SD)  82.0 (15.4)  74.5 (15.6)  80.1 (15.6)  84.8 (17.5)Median (range)    85 (75-95)    78 (60-89)    83 (70-92)    90 (80-95)VAS reduced by ≥ 10 from N/A 26/52 (50) 16/55 (29)  5/42 (12) screening,n/n (%) *EQ-5D visual analogue scale (VAS) assesses overall health on ascale from 0 to 100, with higher scores indicating better health status.EQ-5D, European Quality of Life-5 Dimensions; N/A, not applicable; SD,standard deviation

Sixteen patients (24%) who received CD19 CAR-T cells died, primarilyfrom PD (n=14 [21%]). Two patients had grade 5 AEs (3%), including 1patient with organizing pneumonia related to conditioning chemotherapy,and 1 patient with staphylococcus bacteremia that was related toconditioning chemotherapy and CD19 CAR-T cell treatment.

The median time to peak anti-CD19 CAR T-cell levels was 15 days (range,8-31) after CD19 CAR-T cell infusion and cells were still detectable at24 months in some patients with evaluable samples at the time of datacutoff (6/10 [60%]) in the presence of normal median B-cell levels. CART cell persistence in blood over time as measured by qPCR showed adecline over time in patients with ongoing response and those whorelapsed.

The rapid expansion, resolution to baseline, and clearance over time areconsistent with the known mechanism of action of anti-CD19 CAR T cellsharboring CD28 and CD3ζ costimulatory domains. All 4 patients with noresponse to CD19 CAR-T cell treatment had detectable B-cells atbaseline; none experienced B-cell aplasia at any point on study. Whilethere was no association with baseline tumor burden, expansion wasassociated with response (P=0.0036), with an area under the curve (AUC)and peak that were >200-fold higher among responders vs non-responders,with a similar trend among MRD-negative vs -positive patients at week 4.For both CRS and NE, expansion was greater in patients with grade ≥3 vsthose with grade ≤2 events and the highest peak and AUC were noted inpatients who received tocilizumab ±steroids post-CD19 CAR-T cellinfusion. Median time to peak for evaluated cytokines was 8 days; mostresolved to baseline levels by 28 days. Serum granulocyte-macrophagecolony-stimulating factor and interleukin (IL)-6 were associated withgrade ≥3 CRS and NE. Serum ferritin was associated only with grade ≥3CRS, whereas serum IL-2 and interferon-γ were associated only with grade≥3 NE. In addition, cerebrospinal fluid cytokine analysis revealedhigher levels of C-reactive protein, ferritin, IL-6, IL-8, and vascularcell adhesion molecule 1 in patients with grade ≥3 NE. Induction ofanti-CAR antibodies was not observed in any patient.

Rates of grade ≥3 CRS and NE were similar to those previously reportedwith anti-CD19 CAR T-cell therapies in aggressive NHL. Neelapu S S etal. The New England journal of medicine 2017; 377:2531; Schuster S J etal. The New England journal of medicine 2019; 380:45. There were nodeaths due to CRS or NE, and most symptoms occurred early in treatmentand were generally reversible, with no long-term clinical sequelaeimpairing activities of daily living. The associations observed betweenpeak serum cytokines and Grade ≥3 CRS and/or Grade ≥3 neurologic eventssuggeste a role for CD19 CAR-T cells in these toxicities, given thatthey were observed commensurate with rising and peak levels of CAR Tcells in blood. Associations of peak levels of CAR andmyeloid-cell-related serum cytokines, chemokines, and effector moleculeswith toxicity are consistent with previously published data using asimilar CAR construct in the setting of NHL.^(10,13) One case of grade 4cerebral edema occurred, but the patient fully recovered and remains inCR at 24-months follow-up with no unresolved neurological sequelae.Patient-reported outcomes similarly suggest no long-term quality-of-lifedeficits following CD19 CAR-T cells therapy.

Example 3

This example provided additional analysis to the studies describedabove. Eligible patients (aged ≥18 years) with R/R MCL had an ECOG scoreof 0-1 and ≤5 prior therapies, including chemotherapy, an anti-CD20antibody, and a BTKi. Patients underwent leukapheresis and conditioningchemotherapy (cyclophosphamide 300 mg/m2/d and fludarabine 30 mg/m2/dfor 3 days, on Days −5, −4, −3) followed by a single infusion of CD19CAR-T cells at a target dose of 2×10⁶ CAR T cells/kg, by single IVinfusion on Day 0. The CD19 CAR construct contains a CD3ζ T cellactivation domain and CD28 signaling domain. The manufacturing processremoved circulating CD19-expressing leukemia cells from theleukapheresis product. Sabatino M, et al. Blood 2016; 128:1227.

Some patients received bridging therapy with dexamethasone (20-40 mg orequivalent PO or IV daily for 1-4 days), ibrutinib (560 mg PO daily), oracalabrutinib (100 mg PO twice daily), administered after leukapheresisand completed ≤5 days before initiating conditioning chemotherapy;PET-CT was required post-bridging. The primary endpoint was objectiveresponse rate (ORR [complete response (CR)+partial response]). Keysecondary endpoints were duration of response (DOR), progression-freesurvival (PFS), OS, frequency of adverse events (AEs), levels of CARTcells in blood, and levels of cytokines in serum. Efficacy and safetyanalyses included all patients who received CD19 CAR-T cell therapy.

The key inclusion criteria included R/R MCL defined as diseaseprogression after last regimen or failure to exhibit a CR or PR to thelast regimen; one to five prior therapies that must have included ananthracycline- or bendamustine-containing chemotherapy and anti-CD20monoclonal antibody therapy and Ibrutinib or acalabrutinib; ≥1measurable lesion; age ≥18 years; ECOG of 0 or 1; and adequate bonemarrow, renal, hepatic, pulmonary, and cardiac function. Key exclusioncriteria included prior autologous stem cell transplant (alloSCT); priorCD19-targeted therapy; prior CAR T cell therapy; clinically relevantinfection; and history of or current CNS involvement by MCL or other CNSdisorders.

A total of 68 patients received CD19 CAR-T cell therapy. Presented hereare the updated safety (68 patients) and efficacy (60 patients) results,with median follow-up of 12.3 months [range 7.0-32.3]). A total of 28patients (47%) had ≥24 months follow-up. The median time to initialresponse was 1.0 months [range 0.8-3.1] and to complete response was 3.0months [range 0.9-9.3]. A total of 24 patients (40%) converted fromPR/SD to CR, with 21 (35%) converting from PR to CR and 3 patients (5%)converting from SD to CR.

The median age was 65 years (range, 38-79) and 39 (57%) of patients weremale. One hundred percent (100%) of patients had ECOG score of 0/1, 25%had blastoid morphology, 85% had stage IV disease, 56% hadintermediate/high-risk MIPI, 81% received 3 or more prior therapies,with a median of 3 (range, 1-5) prior therapies, 99% received prioranthracycline or bendamustine, 100% received prior anti-CD20 monoclonalantibody, and 100% received prior BTKi (85% ibrutinib, 24%acalabrutinib, and 9% both). Forty-three (43%) of the patients hadrelapsed after autoSCT, 56% were refractory to ibrutinib, and 12% wererefractory to acalabrutinib. In 34/49 of the patients with availabledata, the Ki-67 index was ≥50%. Twenty-five (37%) of the patientsreceived bridging therapy (21% ibrutinib, 7% acalabrutinib, 18%dexamethasone, 3% methylprednisolone, 9% both BTKi and steroids, 6%ibrutinib and steroid, 3% acalabrutinib and steroid); 23/25 patients hadpost-bridging PET-CT to document measurable disease before CD19 CAR-Tcell infusion (20/23 had an increase in SPD mm² from screening; 3/23 hada slight decrease in SPD mm² from screening).

A high ORR was observed in both efficacy-evaluable and ITT patients. 95%concordance for ORR; 90% concordance for CR. The investigator-assessedORR in 60 efficacy-evaluable patients was 88% (95% CI, 77%-95%) with aCR rate of 70% (95% CI, 57%-81%) and a PR rate of 18% (95% CI, 10%-30%).ORR in 60 efficacy-evaluable patients by IRRC assessment was 93% (95%CI, 84%-98%) with a CR rate of 67% (95% CI, 53%-78%), and a PR rate of27% (95% CI, 16%-40%). ORR was consistent across key subgroups (age, MCLmorphology, Ki-67 index, disease stage, simplified MIPI, steroid use forAE management, tocilizumab use, and bridging therapy use). Theinvestigator-assessed ORR in ITT patients was 80% (95% CI, 69%-88%) witha CR rate of 59% (95% CI, 47%-71%) and a PR rate of 20% (95% CI,12%-31%). ORR in ITT patients by IRRC assessment was 85% (95% CI,75%-92%) with a CR rate of 59% (95% CI, 47%-71%), and a PR rate of 26%(95% CI, 16%-37%).

The median DOR had not been reached after a median follow-up of 12.3months. Fifty-seven percent (57%) of all patients and 78% of patientswith a CR remained in remission. The first 28 patients treated had amedian follow-up of 27.0 months (range, 25.3-32.3), 43% of whichremained in continued remission without additional therapy. Median PFSand median OS were not reached after a median follow-up of 12.3 months.The 12-month PFS rate (95% CI) was 61% (45%-74%). The 12-month OS rate(95% CI) was 83% (71%-91%).

More than 35% of the patients had treatment-emergent adverse events (0%Grade 1; 1% Grade 2; 16% Grade 3; 76% Grade 4, and 3% Grade 5). The mostcommon Grade ≥3 AEs (≥20% of patients) were neutropenia (69%, grade 4),thrombocytopenia (35%, grade 4), anemia (50%, grade 3), hypophosphatemia(22%, grade 3). No patient died from cytokine release syndrome (CRS).Grade ≥3 CRS, assessed by Lee D W, et al. Blood. 2014, 124:188, wasreported in 15% of patients. Most common symptoms of any grade of CRSwere hypotension (51%), hypoxia (34%), and pyrexia (91%). Adverse eventmanagement included tocilizumab (59%) and corticosteroids (22%). Themedian time to onset was 2 days (range 1-13), the median duration was 11days, and 62/62 (100%) of the patients with any grade CRS had resolvedevents.

Neurologic events (NE) of any grade were reported in 63% of patients(31% had Grade ≥3 NE) and included encephalopathy (31%), confusionalstate (21%), and tremor (35%). No patient died from neurologic events.One patient had Grade 4 cerebral edema that fully resolved withaggressive multimodality therapy including ventriculostomy and IVrabbit, anti-thymocyte globulin (ATG). All CRS events and most NE (37/43patients) were reversible. Median time to onset and duration of NE was 7days (range, 1-32) and 12 days, respectively.

A higher peak level of CAR T cells was associated with responders thanwith non-responders (Objective Response). A higher peak level of CAR Tcells was associated with negative than with positive MRD at week 4. Themedian time to peak anti-CD19 CART cell levels after CD19 CAR-T cellinfusion was 15 days (range, 8-31). Anti-CD19 CAR T cells weredetectable at 24 months in most patients with evaluable samples (6/10[60%]). Expansion was associated with response and MRD status. Expansionwas greater in patients with Grade ≥3 vs ≤2 CRS and neurologic events.

Several associations were observed between peak serum biomarker levelsand toxicity. Analytes associated with Grade ≥3 CRS included IL-15, IL-2Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzymeA, granzyme B, and perforin. Analytes associated with Grade ≥3neurologic events included IL-2, IL-1 Ra, IL-6, TNFα, GM-CSF, IL-12p40,IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. And the analytes associatedwith both Grade ≥3 CRS and neurologic events included IL-6, TNFα,GM-CSF, IL-10, MIP-1b, and granzyme B.

The CD19 CAR-T cell treatment described herein, administered in a singleinfusion, showed high rates of durable responses in R/R MCL. The 93%ORR, which included a 67% CR rate, is the highest reported rate ofdisease control in patients with prior BTKi failure. Of the initial 28patients treated, 43% remained in remission after ≥24 months offollow-up. The safety profile is consistent with that reported in priorstudies of anti-CD19 CAR T cell therapies in aggressive NHL No deathsdue to CRS or neurologic events; most symptoms occurred early intreatment and were generally reversible. The efficacy, reliable andrapid manufacturing, and manageable toxicities identify a role for theCD19 CAR-T cell treatment described herein in treating patients with R/RMCL who have an unmet medical need.

Example 4

This example provides additional analysis of the clinical studiesdescribed above. Eligible patients were aged ≥18 years withpathologically confirmed MCL with documentation of either cyclin D1overexpression or presence of t(11; 14), and were relapsed/refractory to1-5 prior regimens for MCL. Prior therapy must have includedanthracycline or bendamustine-containing chemotherapy, an anti-CD20monoclonal antibody, and ibrutinib or acalabrutinib. All patientsreceived prior BTKi. Although patients must have had prior BTKi therapy,it was not required as the last line of therapy before study entry, andpatients were not required to be refractory to BTKi therapy. Eligiblepatients had an absolute lymphocyte count ≥100/μL Patients who underwentautologous SCT within 6 weeks of CD19 CAR-T infusion or had previousCD19-targeted therapy or allogeneic SCT were excluded. All patientsunderwent leukapheresis to obtain cells for CD19 CAR-T cell treatmentmanufacturing. Patients received optional bridging therapy, whichincluded dexamethasone (20-40 mg or equivalent PO or IV daily for 1-4days), ibrutinib (560 mg by mouth (PO) daily), or acalabrutinib (100 mgPO twice daily). The manufacturing process was modified relative to thatof axicabtagene ciloleucel to remove circulating lymphoma cells throughpositive enrichment for CD4⁺/CD8⁺ cells. This product is referred toherein as “the CAR T cells.” This product may also be identified asKTE-X19. Conditioning chemotherapy with fludarabine (30 mg/m²/day) andcyclophosphamide (500 mg/m²/day) was administered on days −5, −4, and −3prior to a single intravenous infusion of 2×10⁶ CAR T cells/kg of CD19CAR-T cells on day 0. More details about patient treatment can be foundin Example 2.

The goals of this study were two-fold. First, to compare thepharmacological profile of the CAR T product in lower- and higher-riskpatients in the clinical trial ZUMA-2, defined by TP53, (tumor proteinp53) gene mutation status and Ki-67 tumor proliferation index. Patientswith high-risk MCL characteristics, including tumor protein p53 gene(TP53) mutation and high Ki-67 proliferation index, typically have apoor prognosis with current standard therapies. Cheah C Y, et al. J ClinOncol. 2016; 34:1256-1269. Lower-risk patients in this analysis had aKi-67 proliferation index <50% (by central evaluation) or wild-typeTP53; higher-risk patients had Ki-67 ≥50% or TP53 mutation by nextgeneration sequencing. In the primary efficacy analysis of ZUMA-2(N=60), the ORR was 93% (67% CR) after a median follow-up of 12.3months. 57% of all patients and 78% of patients in CR had ongoingresponses. The ORR was generally comparable between lower- andhigher-risk patients in ZUMA-2, including in patients with Ki-67proliferation index < or ≥50% and unmutated vs mutated TP53. Wang M, etal. New Engl J Med. 2020; 382:1331-1342.

The second goal was to characterize the pharmacodynamic profile inpatients who achieved early (Day 28) Minimal residual disease(MRD)-negative status and those with Grade 4 neurotoxicity. In aprevious analysis of ZUMA-2 results, CAR T cell levels in blood by peakand area under the curve (AUC) on Days 0-28 were associated with ORR(including undetectable MRD) and Grade ≥3 CRS and neurologic events.Wang M, et al. New Engl J Med. 2020; 382:1331-1342. In that analysis,CRS and neurologic events were mostly reversible (N=68 treatedpatients): 15% had Grade ≥3 CRS; 31% had Grade ≥3 neurologic events; andtwo had Grade 5 AEs (one of which was CAR T product-related). MRD (10⁻⁵sensitivity) was assessed by next-generation sequencing, as previouslyreported. Wang M, et al. New Engl J Med. 2020; 382:1331-1342.

This update reports pharmacology data for all 68 patients in ZUMA-2 whowere treated with the CAR T cells Product attributes, CAR T cell levelsin blood, and cytokine levels in serum, and their associations withclinical outcomes, were analyzed by using previously described methods.Locke Fla., et al. Mol Ther. 2017; 25:285-295. Wilcoxon rank-sum testwas used to measure associations between subgroup outcomes and CAR Tcell and cytokine levels. P values were not adjusted for multipletesting.

CAR T cell product attributes were generally comparable acrossprognostic groups defined by Ki-67 proliferation index and TP53 mutationstatus. There was a trend toward more differentiated phenotypes in thehigh-Ki-67 subgroup, and CD4-based phenotypes in patients with TP53mutation. (Table 8).

TABLE 8 Treated Ki-67 Proliferation Index TP53 Median Patients^(a) <50%≥50% Mutation Nonmutation (range) (n = 65) (n = 14) (n = 34) (n = 6) (n= 30) CD4/CD8 0.7 0.8 0.7 1.2 0.7 Ratio (0.04, 3.7) (0.4, 1-7) (0.04,3.7) (0.7, 3.7) (0.04, 1.9) Naive T 24.5 30.4 20.1 23.0 25.2 cells, %(0.3, 80.7) (11.0, 57.0) (0.3, 68.8) (11.8, 46.5) (0.3, 78.1) Central12.8 10.1 12.0 13.2 10.2 memory T (2.3, 51.6) (8.4, 45.0) (2.3, 51.6)(6.0, 51.6) (2.3, 45.0) cells, % Effector 24.5 19.4 29.1 25.9 29.4memory T (0.8, 70.3) (6.3, 56.1) (5.8, 70.3) (7.0, 38.2) (2.2, 70.3cells, % Effector T 28.7 23.7 32.4 29.1 29.1 cells, % (2.8, 65.2) (11.5,49.30) (2.8, 65.2) (2.8, 44.7) (8.4, 54.5) ^(a)Of all 68 treatedpatients, product characteristic data were available for 65 totalpatients. Product characteristic data were available for 48/49 totalpatients with Ki-67 data available and for all 36 patients with TP53mutation data available. TP53, tumor protein p53 gene

There was also comparable CAR T cell expansion in groups with differentprognostic factors defined by Ki-67 proliferation index and TP53mutation status. Both peak levels and AUC of CAR T cells in the bloodafter administration were comparable in patients with wild-type vsmutated TP53 or Ki-67 proliferation index <50% vs ≥50%, which wasconsistent with the comparable efficacy in these subgroups. The primaryendpoint of objective response rate (ORR) in patients was shown in Table9. The median time to response was 28 days (range: 24 to 92 days) with amedian follow-up time of 12.3 months. 28 patients had potentialfollow-up for ≥24 months and 12 of these patients remained in remission.Efficacy was established based on complete response and duration ofresponse (DOR).

The ORR was 100% vs 94% in patients with Ki-67 proliferation index <50%vs ≥50% whereas the CR rate was 64% vs 78% in patients Ki-67proliferation index <50% vs ≥50%. Table 9. The number of patients withavailable data for Ki-67 proliferation index was 49.

TABLE 9 ORR (95% CI), % CR Rate (95% CI), % Ki-67 PI < 50% 100 (77-100)64 (35-87) Ki-67 PI ≥ 50% 94 (79-99) 78 (60-91)

The ORR was 100% for both in patients with wild-type vs mutated TP53whereas the CR rate was 67% vs 100% in wild-type vs mutated TP53. Table10. The number of patients with available data for TP53 was 36. All sixpatients with TP53 mutation and all 30 patients with no mutationresponded. Among the six patients with TP53 mutation, three had Grade ≥3neurotoxicity and two had Grade ≥3 CRS

TABLE 10 ORR (95% CI), % CR Rate (95% CI), % TP53 mutation 100 (54-100)100 (54-100) TP53 nonmutation 100 (88-100) 67 (47-83)

Up to 44 biomarkers in serum were measured pretreatment, at Day 0, andat various time points through Day 28 post CAR T cell infusion,including IL (interleukins); INF-γ (interferon gamma), MCP-1 (monocytechemoattractant protein-1), IL-2Rα (IL-2 receptor alpha), sPD-L1(soluble programmed death-ligand 1) and sVCAM (soluble vascular celladhesion molecule). The pharmacodynamic profile for the two prognosticgroups with Ki-67 proliferation index <50% vs ≥50% was comparable withregard to proliferative (IL-15, IL-2), inflammatory (IL-6, IL-2Rα,sPD-L1 and VCAM-1), immune-modulating (IFN-γ, IL-10), chemokine (IL-8and MCP-1)), and effector cytokines (Granzyme B). In addition, there wasa trend for increased proliferative (IL-15, IL-2) and inflammatory(IL-6, IL-2Rα, sPD-L1 and VCAM-1) cytokine levels in patients withmutated TP53 vs wild-type TP53. FIGS. 1A-1F.

There was also an increase in the peak levels of select cytokines inserum among patients who achieved MRD-negative status. MRD was analyzedin 29 of 68 patients (43%); 24 of these patients (83% [19 patients witha complete response and 5 with a partial response]) were MRD negative atone month post CAR T cell administration. At one month post CAR T celladministration, MRD negative (n=24/29) vs -positive patients (n=5/29)had increased median peak levels of interferon (IFN)-γ and interleukin(IL)-6 and a trend towards increased IL-2. Cytokine levels peaked inserum within 7 days of treatment. Consistent trends were seen for PD-L1and Granzyme B. Increased peak CAR T cell levels, measured within 14days posttreatment, were also seen in patients who were MRD negative at1 month. FIGS. 2A-2I.

Six patients developed Grade 4 neurologic events, including one withcerebral edema. Three patients had concurrent Grade 4 CRS. Patients withGrade 4 neurologic events showed increased peak levels ofproinflammatory serum biomarkers (e.g., IFNγ, MCP-1, TNF-α, IL-2 andIL-6) compared to patients without neurologic events.

The cerebral edema was completely resolved following aggressivemultimodality therapy. Wang M, et al. New Engl J Med. 2020;382:1331-1342. Expansion of CART cells and peak serum levels of IL-2were highest in this patient; the rise in multiple cytokines wasseveral-fold higher in this patient compared with the median of otherstudy/ZUMA-2 patients. Table 11.

TABLE 11 Patient With Other ZUMA-2 Patients Cerebral Edema (n = 67),Median (IQR) Peak (Post CAR T Peak Baseline cell Baseline (Post CAR Tcell (Day 0) administration) (Day 0) administration) CAR T cell levels,0 431.3 0  83.1 (17.2-264.3)^(a) cells/μL IFN-γ, pg/mL 7.5 584.4   7.5(7.5-17.7)  411.2 (144.8-1876) MCP-1, pg/mL 462.6 1500  882.9 (557.2-1084.3 (804.2-1500) 1164.8) TNFα, pg/mL 1.9 10.4   5.7 (3.2-10.6)   9.5(5.5-23.2) sVCAM-1, ng/mL 527.5 1659.7 1195.9 (791.7- 1900.7 (1032.4-2533.1) 3646.7) IL-2, ng/mL 0.9 16.7   0.9 (0.9-0.9)   6.0 (3.0-14.4)IL-6, pg/mL 1.6 159.5   1.6 (1.6-6.4)   87.9 (12.9-879.1) CRP, mg/L 6.818.2   30.5 (15.1-63.0)  119.4 (54.6-173.8) Ferritin, ng/mL 606.3 824.2 502.4 (273.5-877.7)   1265 (597.8-2970.1) IL-15, ng/mL 29.1 56.1   33.2(25.4-48)   38.4 (29.7-61.7) ^(a)Out of 66 patients with available data.

CAR T cell pharmacokinetic and pharmacodynamic profiles were comparableacross MCL patient groups with different prognostic marker statusassociated with lower and higher risk (defined by Ki-67 and mutatedTP53), consistent with comparable clinical response rates. There was atrend toward higher levels of proinflammatory markers in patients withmutated TP53.

The pharmacodynamic profile of CAR T cell administration was associatedwith efficacy (MRD status at 1 month) and Grade 4 treatment-emergentneurologic events. The patient who developed cerebral edema had thehighest peak CAR T cell levels and serum IL-2, as well as elevatedproinflammatory markers posttreatment.

Example 5

A Phase 2 single-arm clinical study was conducted for a CD19-directedgenetically modified autologous T cell immunotherapy treating patientswith relapsed or refractory mantle cell lymphoma (MCL) who had receivedone or more prior treatment (which may have included an anti-CD20antibody, anthracycline- or bendamustine-containing chemotherapy, and/ora Bruton tyrosine kinase inhibitor (BTKi) such as ibrutinib oracalabrutinib). Eligible patients also had disease progression aftertheir last treatment or refractory disease to their most recenttreatment. The study excluded patients with active or seriousinfections, prior allogeneic hematopoietic stem cell transplant (HSCT),detectable cerebrospinal fluid malignant cells or brain metastases, andany history of central nervous system (CNS) lymphoma or CNS disorders.

Patient's peripheral blood mononuclear cells were obtained via aleukapheresis procedure. The mononuclear cells were enriched for T cellsby selection for CD4+ and CD8+ cells, activated with anti-CD3 andanti-CD28 antibodies in the presence of IL-2, then transduced with areplication-incompetent viral vector containing FMC63-28Z CAR, achimeric antigen receptor (CAR) comprising an anti-CD19 single-chainvariable fragment (scFv), CD28 and CD3-zeta domains. Without being boundto any hypothesis, selection for CD4+ and CD8+ cells may have reducedpotential circulating CD19-expressing tumor cells in patients'leukapheresis material to be included during the ex vivo manufacturingprocess. The T cell product of this process may be identified asKTE-X19. The anti-CD19 CAR T cells were expanded, washed, formulatedinto a suspension, and cryopreserved. Prior to receiving the anti-CD19CAR T cell therapy, patients were treated with a lymphodepletingchemotherapy regimen of cyclophosphamide 500 mg/m² intravenously andfludarabine 30 mg/m² intravenously on each of the fifth, fourth, andthird days before infusion of CAR T-cells; patients may also havereceived acetaminophen and diphenhydramine or another H1-antihistamineapproximately 30 to 60 minutes prior to infusion of anti-CD19 CAR Tcells. Prophylactic use of systemic corticosteroids was avoided as itmay interfere with the activity of CAR T cells.

The target dose was 2×10⁶ CAR positive viable T cells or anti-CD19 CAR Tcells per kg body weight, with a maximum of 2×10⁸ anti-CD19 CAR T cells(for patients 100 kg and above) cells. 68 patients received a singleinfusion (by either gravity or a peristaltic pump for approximately 30minute) of anti-CD19 CAR T cells, and 60 of these patients were followedfor at least 6 months after their Week 4 disease assessment, qualifyingthem as efficacy-evaluable. 56 patients received 2×10⁶ anti-CD19 CAR Tcells/kg; 1 patient received a dose of 1×10⁶ anti-CD19 CAR T cells/kg, 1patient received a dose of 1.6×10⁶ anti-CD19 CAR T cells/kg, 2 patientsreceived a dose of 1.8×10⁶ anti-CD19 CART cells/kg, and −2 patientsreceived a dose of 1.9×10⁶ anti-CD19 CART cells/kg. Of these 60patients, the median age was 65 years (range: 38 to 79 years), 51 weremale, and 56 were white. 50 patients had stage IV disease. Based on thesimplified Mantle Cell Lymphoma International Prognostic Index (s-MIPI),25 patients were classified as low risk, 25 patients were classified asintermediate risk, 8 patients was classified as high risk, and 2patients had an unknown risk status. 20 patients had baseline bonemarrow examinations performed per protocol; of these, 10 were negative,8 were positive, and 2 were indeterminate. The median number of priortherapies among all 60 efficacy-evaluable patients was 3 (range: two tofive). 26 patients had relapsed after or were refractory to autologousHSCT. 21 patients had relapsed after their last therapy for MCL, while36 patients were refractory to their last therapy for MCL. 14 patientshad blastoid MCL. Following leukapheuresis and prior to infusion ofanti-CD19 CAR T cells, 21 patients received bridging therapy. 19 weretreated with a BTKi, 14 patients were treated with corticosteroid, and 6patients were treated with both a BTKi and a corticosteroid. 53 patientsreceived a lymphodepleting chemotherapy regimen of cyclophosphamide 500mg/m² intravenously and fludarabine 30 mg/m² intravenously, both givenon each of the fifth, fourth, and third days prior to anti-CD19 CAR Ttherapy (day 0). The remaining 7 patients received the same dosage oflymphodepleting chemotherapy over 4 or more days prior to CAR T therapy.The primary endpoint of objective response rate (ORR) in patients wasshown in Table 12. The median time to response was 28 days (range: 24 to92 days) with a median follow-up time of 12.3 months. Twenty-eightpatients had potential follow-up for ≥24 months and twelve of thesepatients remained in remission. Efficacy was established based oncomplete response and duration of response (DOR).

TABLE 12 Efficacy-Evaluable Leukapheresed Patients Patients N = 60 N =74 Response Rate Objective Response Rate 52 59 (ORR) [75, 94] [69, 88][95% CI] Complete Remission (CR) 37 41 Rate [48, 74] [43, 67] [95% CI]Partial Remission (PR) 15 18 Rate [95% CI] [15, 38] [15, 36] Duration ofResponse (DOR) ^(a) Median in months [95% NR [8.6, NE] NR [11.8, NE] CI]Range in months 0.0^(b), 29.2^(b) 0.0^(b), 29.2^(b) DOR, if bestresponse is NR [13.6, NE] NR [13.6, NE] CR, median in months 1.9^(b),29.2^(b) 0.0^(b), 29.2^(b) [95% CI] Range in months DOR, if bestresponse is 2.2 [1.5, 5.1] 4.2 [1.5, 5.1] PR, median in days [95%0.0^(b), 22.1^(b) 0.0^(b), 22.1^(b) CI] Range in months Median Follow-upfor 8.6 8.1 DOR in months CI, confidence interval; NE, not estimable;NR, not reached; PR, partial remission. ^(a) Among all responders. DORis measured from the date of first objective response to the date ofprogression or death. ^(b)A censored value.CRS (cytokine release syndrome) was observed in 75 out of 82 patients,including ≥Grade 3 (Lee grading system1) CRS in 15 out of 82 patients.The median time to onset of CRS was 3 days (range: 1 to 13 days) and themedian duration of CRS was 10 days (range: 1 to 50 days). Among patientswith CRS, key manifestations (i.e., manifestations that occurred in >10%of the patients) included fever (99% of the patients), hypotension (60%of the patients), hypoxia (37% of the patients), chills (33% of thepatients), tachycardia (37% of the patients), headache (24% of thepatients), fatigue (19% of the patients), nausea (13% of the patients),alanine aminotransferase increased (13% of the patients), aspartateaminotransferase increased (12% of the patients), and diarrhea (11% ofthe patients). Serious events associated with CRS included hypotension,fever, hypoxia, acute kidney injury, and tachycardia. In response toCRS, patients may have received tocilizumab and/or corticosteroids perthe indications in Table 13.

TABLE 13 CRS Grade^(a) Tocilizumab Corticosteroids Grade 1 If notimproving after 24 hours, Not applicable. Symptoms require administertocilizumab^(c) 8 symptomatic treatment only mg/kg intravenously over 1(e.g., fever, nausea, fatigue, hour (not to exceed 800 mg). headache,myalgia, malaise). (for other cancers, this may be not applicable) Grade2 Administer tocilizumab 8 Manage per Grade 3 if no Symptoms require andmg/kg intravenously over 1 improvement within 24 respond to moderatehour (not to exceed 800 mg). hours after starting intervention. Repeattocilizumab every 8 hours tocilizumab. Oxygen requirement less than asneeded if not responsive to If improving, taper 40% FiO₂ or hypotensionintravenous fluids or increasing corticosteroids. responsive to fluidsor low supplemental oxygen. Limit to a dose of one vasopressor ormaximum of 3 doses in a 24-hour Grade 2 organ toxicity^(b). period;maximum total of 4 doses if no clinical improvement in the signs andsymptoms of CRS. If improving, discontinue tocilizumab. Grade 3 PerGrade 2 Administer Symptoms require and methylprednisolone 1 respond toaggressive mg/kg intravenously twice intervention. daily or equivalentOxygen requirement greater dexamethasone (e.g., 10 than or equal to 40%FiO₂ or mg intravenously every 6 hypotension requiring high- hours)until Grade 1, then dose or multiple taper corticosteroids. vasopressorsor Grade 3 If improving, manage as organ toxicity or Grade 4 Grade 2.transaminitis. If not improving, manage as Grade 4. Grade 4 Per Grade 2Administer Life-threatening symptoms. methylprednisolone Requirementsfor ventilator 1000 mg intravenously support or continuous veno- per dayfor 3 days. venous hemodialysis If improving, taper (CVVHD), or Grade 4organ corticosteroids, and toxicity (excluding manage as Grade 3.transaminitis). If not improving, consider alternate immunosuppressants.^(a)Lee DW et al (2014). Current concepts in the diagnosis andmanagement of cytokine release syndrome. Blood. 2014 Jul 10; 124(2):188-195. ^(b)Refer to Table 14 for management of neurologic toxicity.^(c)Refer to tocilizumab Prescribing Information for details

Neurologic events were observed in 53 patients, 20 of whom experiencedGrade 3 or higher (severe or life-threatening) adverse reactions. Themedian time to onset for neurologic events was 6 days (range: 1 to 32days). Neurologic events were resolved for 52 out of 66 patients with amedian duration of 21 days (range: 2 to 454 days). 3 patients hadongoing neurologic events at the time of death, including 1 patient withserious encephalopathy. The remaining unresolved neurologic events wereeither Grade 1 or Grade 2. 54 patients experienced CRS by the onset ofneurological events. 5 patients did not experience CRS with neurologicevents and 8 patients developed neurological events after the resolutionof CRS. 56 patients experienced the first CRS or neurological eventwithin the 7 seven days after infusion of anti-CD19 CAR T cells.

The most common neurologic events (occurring in >10% of the patients)included encephalopathy (51% of the patients), headache (35% of thepatients), tremor (38 of the patients aphasia (23% of the patients), anddelirium (16% of the patients). Serious events including encephalopathy,aphasia, and seizures occurred after treatment. Some adverse reactionsobserved in at least ten percent of treated patients included: Blood andLymphatic System Disorders (Coagulopathy, Cardiac Disorders,Tachycardias, Bradycardias, Non-ventricular Arrhythmias);Gastrointestinal Disorders (Nausea, Constipation, Diarrhea, Abdominalpain, Oral pain, Vomiting, Dysphagia); General Disorders andAdministration Site Conditions (Pyrexia, Fatigue, Chills, Edema, Pain);Immune System Disorders (Cytokine release syndrome,Hypogammaglobulinemia); Infections and Infestations (Infection—pathogenunspecified, Viral infections, Bacterial infections); Metabolism andnutrition disorders (Decreased appetite), Musculoskeletal and ConnectiveTissue Disorders (Musculoskeletal pain, Motor dysfunction); NervousSystem Disorders (Encephalopathy, Tremor; Headache, Aphasia, Dizziness,Neuropathy); Psychiatric Disorders (Insomnia, Delirium, Anxiety); Renaland Urinary Disorders (Renal insufficiency, Urine output decreased);Respiratory, Thoracic and Mediastinal Disorders (Hypoxia, Cough,Dyspnea, Pleural effusion); Skin and Subcutaneous Tissue Disorders(Rash); and Vascular Disorders (Hypotension, Hypertension, Thrombosis).Patients who experience Grade 2 or higher neurologic toxicities may havebeen treated per the indications shown in Table 14.

TABLE 14 Grading Assessment^(a) Concurrent CRS No Concurrent CRS Grade 2Administer tocilizumab per Table Administer dexamethasone 10 13 formanagement of Grade 2 mg intravenously every 6 hours CRS. until theevent is Grade 1 or less, If not improving within 24 hours after thentaper corticosteroids. starting tocilizumab, administer dexamethasone 10mg intravenously every 6 hours until the event is Grade 1 or less, thentaper corticosteroids. If still not improving, manage as Grade 3.Consider non-sedating anti-seizure medicines (e.g., levetiracetam) forseizure prophylaxis. Grade 3 Administer tocilizumab per Table Administer13 for management of Grade 2 dexamethasone 10 mg CRS. intravenouslyevery 6 In addition, administer dexamethasone 10 hours. mg intravenouslywith the first dose of Continue dexamethasone use tocilizumab and repeatdexamethasone until the event is Grade 1 or less, dose every 6 hours.Continue then taper corticosteroids. dexamethasone use until the eventis If not improving, manage as Grade Grade 1 or less, then taper 4.corticosteroids. If improving, discontinue tocilizumab and manage asGrade 2. If still not improving, manage as Grade 4 (below). Considernon-sedating anti-seizure medicines (e.g., levetiracetam) for seizureprophylaxis. Grade 4 Administer tocilizumab per Table Administer 13 formanagement of Grade 2 methylprednisolone 1000 mg CRS. intravenously perday for 3 Administer methylprednisolone 1000 mg days. intravenously perday with first dose of If improving, then manage as tocilizumab andcontinue Grade 3. methylprednisolone 1000 mg If not improving,intravenously per day for 2 more days. consider alternate If improving,then manage as Grade 3. immunosuppressants. If not improving, consideralternate immunosuppressants. [A1] Consider non-sedating anti-seizuremedicines (e.g., levetiracetam) for seizure prophylaxis. ^(a)Severitybased on Common Terminology Criteria for Adverse Events.

After infusion of anti-CD19 CAR T cells, pharmacodynamic responses wereevaluated over a four-week interval by measuring transient elevation ofcytokines, chemokines and other molecules in blood. Levels of IL-6,IL-8, IL-10, IL-15, TNF-α, IFN-γ, and/or sIL2Rα were analyzed. Peakelevation of these cytokine levels was generally observed between 4 and8 days after infusion, and levels generally returned to baseline within28 days. A period of B cell aplasia was expected. Following infusion, aninitial expansion of anti-CD19 CAR T cells was followed by a decline tonear baseline levels by 3 months. Peak levels of anti-CD19 CAR T cellsoccurred within the first 7 to 15 days after infusion. Results showedthat the levels of anti-CD19 CAR T cells in blood were associated withobjective response (i.e. complete remission (CR) or partial remission(PR)). The median peak anti-CD19 CAR T cell level in responders (thosewith complete remission and partial remission) was 102.4 cells/μL(range: 0.2 to 2589.5 cells/μL; n=51), and in nonresponders was 12.0cells/μL (range: 0.2 to 1364.0 cells/μL, n=8). The median AUC Day 0-28(AUC₀₋₂₈) in patients with an objective response was 1487.0cells/μL·days (range: 3.8 to 2.77×10⁴ cells/μL·days; n=51) and 169.5cells/μL·days in nonresponders (range: 1.8 to 1.17 10×10⁴ cells/μL·days;n=8). The median peak (24.7 cells/μL) anti-CD19 CAR T cell (peak: andAUC₀₋₂₈ levels (360.4 cells/μL·days) in patients (n=18) who receivedneither corticosteroids nor tocilizumab was similar to those of patients(n=2) who received only corticosteroids (peak: 24.2 cells/μL; AUC₀₋₂₈:367.8 cells/μL·days). In the patients who received only tocilizumab(n=10), the mean peak anti-CD19 CART cells was 86.5 cells/μL and AUC₀₋₂₈was 1188.9 cells/μL·days. In the patients who received bothcorticosteroids and tocilizumab (n=37), the mean peak was 167.2 cells/μLand AUC₀₋₂₈ was 1996.0 cells/μL·days. The median peak anti-CD19 CART-cell values were 74.1 cells/μL in patients ≥65 years of age (n=39) and112.5 cells/μL in patients <65 years of age (n=28). Median anti-CD19 CART-cell AUC₀₋₂₈ values were 876.5 cells/μL·day in patients ≥65 years ofage and 1640.2 cells/μL·day in patients <65 years of age. Gender had nosignificant impact on AUC₀₋₂₈ and C_(max) of anti-CD19 CAR T cells.

Example 6

Patients with MCL who progress after BTKi therapy have a median overallsurvival of only 5.8 months with salvage therapies. ZUMA-2(ClinicalTrials.gov Identifier: NCT02601313) is a Phase 2,registrational, multicenter study of patients with R/R MCL after 1-5prior therapies, including a BTKi. Patients were administered anautologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy,prepared and administered as described in EXAMPLE 5. This anti-CD19 CART cell product may be referred to as KTE-X19. In the primary analysis ofZUMA-2 (N=60), the objective response rate (ORR) with anti-CD19 CAR Tcell treatment (median follow-up 12.3 months) was 93% (67% completeresponse [CR] rate). This Example describes a comparative analysis ofthe pharmacology profile of the anti-CD19 CAR T cell treatment preparedas described in EXAMPLE 5 and outcomes by MCL morphology and prior BTKiexposure (ibrutinib [Ibr] and/or acalabrutinib [Acala]), accompanied bybasic product attribute characterization.

Eligible patients with R/R MCL underwent leukapheresis and conditioningchemotherapy followed by a single infusion of 2×10⁶ anti-CD19 CAR Tcells/kg. Product attributes (e.g., IFNγ production by the anti-CD19CART cells upon co-culture with CD19+ cells), CAR T cell levels inblood, and cytokine levels in serum were assessed using methodspreviously described (see previous EXAMPLES). Clinical outcomes arereported in the 60 efficacy-evaluable patients; product attributes andpharmacology data are reported for all 68 treated patients.

At baseline, 40 patients (59%) had classical MCL, 17 (25%) had blastoidMCL, and 4 (6%) had pleomorphic MCL, as assessed by investigator. Beforestudy entry, 52 patients (76%) had prior Ibr, 10 (15%) had prior Acala,and 6 (9%) had both; 88% had BTKi-refractory disease. In themanufactured anti-CD19 CAR T product, median (range) CD4+/CD8+ T cellratios for patients with classical, blastoid, or pleomorphic MCL were0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0), respectively. Product Tcell phenotypes (median [range]) included less differentiated CCR7+ Tcells (classical 40.0% [2.6-88.8]; blastoid 35.3% [14.3-73.4];pleomorphic 80.8% [57.3-88.8]) and effector and effector memory CCR7− Tcells (classical 59.9% [11.1-97.4]; blastoid 64.8% [26.6-85.7];pleomorphic 19.2% [11.1-42.7]). Median (range) interferon (IFN)-γ levelsby coculture in patients with classical, blastoid, or pleomorphic MCLwere 6309.5 pg/mL (424.0-20,000), 6510.0 pg/mL (2709.0-18,000), or7687.5 pg/mL (424.0-12,000), respectively. In patients with classical,blastoid, or pleomorphic MCL, median (range) peak CAR T cell levels were77.6 cells/μL (0.2-2241.6), 35.0 cells/μL (0.2-2589.5), or 144.9cells/μL (39.2-431.3), respectively. ORR/CR rates were 93%/65% inpatients with classical MCL, 88%/53% in those with blastoid MCL, and100%/75% in those with pleomorphic MCL. The 12-mo survival rates inpatients with classical, blastoid, or pleomorphic MCL were 86.7%, 67.9%,or 100%, respectively. Grade ≥3 cytokine release syndrome (CRS) andneurologic events occurred in 15% and 38% of patients with classicalMCL, 6% and 8% of patients with blastoid MCL, and 25% and 50% ofpatients with pleomorphic MCL.

For patients who received prior Ibr, Acala, or both, median CD4+/CD8+ Tcell ratios in the manufactured anti-CD19 CART cell product were 0.7(range, 0.04-3.7), 0.6 (range, 0.3-1.2), or 1.0 (range, 0.7-1.9),respectively. Product T cell phenotypes (median [range]) included lessdifferentiated CCR7+ T cells (Ibr 39.3% [2.6-86.4]; Acala 42.7%[16.3-88.8]; both 49.5% [14.3-83.0]) and CCR7− effector and effectormemory T cells (Ibr 60.6 [13.7-97.4]; Acala 57.3% [11.1-83.8]; both50.6% [17.0-85.7]). Median (range) levels of IFN-γ by coculture inpatients with prior Ibr, Acala, or both was 6496.0 pg/mL (424.0-20,000),5972.5 pg/mL (2502.0-18,000), or 7985.5 pg/mL (2709.0-12,000),respectively. For patients with prior Ibr, Acala, or both, median(range) peak CART cell levels were 95.9 (0.4-2589.5), 13.7 (0.2-182.4),or 115.9 (17.2-1753.6), respectively. ORR/CR rates were 94%/65% inpatients with prior Ibr, 80%/40% in patients with prior Acala, and100%/100% in patients with both BTKis. The 12-month survival rates inpatients with prior Ibr, Acala, or both were 81%, 80%, or 100%,respectively. Grade ≥3 CRS and neurologic events occurred in 17% and 31%of patients with prior Ibr, 10% and 10% of patients with Acala, and 0and 67% of patients with both BTKis. While post-treatment CAR T celllevels were lower in patients with blastoid morphology or previouslytreated with Acala alone, mirrored by similar trends in clinicaloutcomes, all subgroups defined by MCL morphology or prior BTKi drewclinical benefit from anti-CD19 CAR T cell treatment.

Example 7

This EXAMPLE provides an updated analysis of efficacy, safety, andpharmacology for all patients in ZUMA-2 with a minimum follow-up of 1year. Eligible patients with R/R MCL underwent leukapheresis andconditioning chemotherapy followed by a single infusion of anti-CD19 CART cell therapy (2×10⁶ CAR T cells/kg) as described in the previousEXAMPLES. The primary endpoint was ORR (CR+partial response) as assessedby an Independent Review Committee according to the LuganoClassification. Efficacy data are reported for the 60 treated patientswith ≥1 year of follow-up; safety data are presented for all 68 treatedpatients.

The median follow-up was 17.5 months (range, 12.3-37.6). The ORR was 92%(95% CI, 81.6-97.2), with a CR rate of 67% (95% CI, 53.3-78.3). Of allefficacy-evaluable patients, 48% had ongoing responses as of the datacutoff. Medians were not reached for duration of response,progression-free survival (PFS), or overall survival; 15-month estimateswere 58.6% (95% CI, 42.5-71.7), 59.2% (95% CI, 44.6-71.2), or 76.0% (95%CI, 62.8-85.1), respectively. In patients who achieved a CR, the medianPFS was not reached (15-month rate, 75.1% [95% CI, 56.8-86.5]); in thosewho achieved a partial response, the median PFS was 3.1 months (95% CI,2.3-5.2). Median PFS was 1.1 months (95% CI, 0.9-3.0) in nonrespondingpatients. The first 28 patients treated had a median follow-up of 32.3months (range, 30.6-37.6); 39.3% of these patients remained in continuedremission with no further therapy.

Common grade ≥3 adverse events were neutropenia (85%), thrombocytopenia(53%), anemia (53%), and infections (34%). Grade ≥3 cytopenias werereported in 60% of patients ≥30 days post-infusion. Grade ≥3 cytokinerelease syndrome occurred in 15% of patients; 59% received tocilizumabfor management of CRS. Grade ≥3 neurologic events (NEs) were reported in31% of patients, and 38% received steroids for NE management. All CRSevents and most NEs (37/43) resolved. There were no Grade 5 CRS eventsor NEs, and no new Grade 5 events occurred with additional follow-up.There were 2 cases of Grade 2 cytomegalovirus infection, 1 case each ofGrade ≥3 hypogammaglobulinemia and Grade ≥3 tumor lysis syndrome, and nocases of Epstein-Ban virus-associated lymphoproliferation,replication-competent retrovirus, hemophagocytic lymphohistiocytosis, oranti-CD19 CAR T-cell-related secondary cancers.

Median peak CAR T cell levels and median area under the curve (Days0-28) were 98.9 cells/μL (range, 0.2-2565.8) and 1394.9 cells/μL (range,3.8-27,700) in patients with ongoing responses at 12 months, 202.6cells/μL (range, 1.6-2589.5) and 2312.3 cells/μL (range, 19.0-27,200) inpatients who were relapsed at 12 months, and 0.4 cells/μL (range,0.2-95.9) and 5.5 cells/μL (range, 1.8-1089.1) in nonresponders. Of the57 efficacy-evaluable patients with data available, 84% had B cellsdetectable by flow cytometry at baseline. Of those in ongoing responsesat 12 months, 10 of 26 patients (38%) with evaluable samples had B cellsdetectable at 3 months, and 10 of 18 (56%) had detectable B cells at 12months; gene-marked CAR T cells were no longer detectable at 12 monthsin 5 of 28 evaluable patients (17%). The ZUMA-2 study continues to showa substantial and durable clinical benefit of anti-CD19 CAR T celltherapy with manageable safety in patients with R/R MCL. Within thispatient population, which lacked curative treatment options, mostpatients achieved durable CR, and no new safety signals were reported.Although early CAR T cell expansion was higher in patients who achievedan objective response, those who later relapsed showed elevated CAR Tcell levels pointing to alternate mechanisms of secondary treatmentfailure in MCL.

Example 8

Although approximately 80-85% of patients with ALL achieve durablecomplete remissions (CRs) after initial treatment, the remaining 15-20%of patients with relapsed or refractory (R/R) ALL have unfavorableoutcomes with a 2-year event-free survival of ≤40% in patients withrelapsed disease. An anti-CD19 CAR T cell therapy prepared as describedabove shown high rates of complete responses, with a manageable safetyprofile for adult patients with R/R B cell lymphoma (see previousEXAMPLES). In particular se, e.g., Example 5). ZUMA-4(ClinicalTrials.gov Identifier: NCT02625480) is a Phase 1/2 studyevaluating this anti-CD19 CAR T cell therapy in pediatric and adolescentpatients with R/R B cell ALL or NHL. End-of-Phase 1 interim analysis ofZUMA-4 showed the feasibility of anti-CD19 CAR T cell therapy withoptimized dosing and adverse event (AE) management strategies for thetreatment of pediatric patients with R/R ALL. The protocol for Phase 2of ZUMA-4 has been amended to include broader B cell ALL enrollmentcriteria with a focus on patients with early relapse associated withpoorer outcomes, and an NHL cohort was added.

Key B cell ALL enrollment criteria included age ≤21 years, weight ≥10kg, and B cell ALL that was primary refractory, relapsed within 18months of first diagnosis, R/R after ≥2 lines of systemic therapy, orR/R after allogeneic stem cell transplantation at least 100 days priorto enrollment. The B cell ALL was also B-precursor cell ALL R/R afterautologous stem cell transplantation at least 100 days prior toenrollment and off immunosuppressive medications for ≥4 weeks. TheLansky (age <16 years) or Karnofsky (age ≥16 years) performance statuswas PS ≥80, weight ≥6 kg. Eligible patients included patients with CNS-1disease, patients with CNS-2 disease without clinically evidentneurological changes, and patients with >5% BM blasts or MRD-positivedisease (threshold 10⁻⁴ by flow or PCR). CNS-1 disease was defined by nodetectable lymphoblasts in CSF; CNS-2 disease was defined by detectabledisease and white blood cell count <5/μL in CSF. CNS-3 disease wasdefined by WBC ≥5/μL in CSF. Criteria for disease burden had beenamended to also include patients with minimal residual disease-positivedisease at enrollment. Patients with Philadelphia chromosome-positiveALL were eligible if intolerant to tyrosine kinase inhibitor therapy orif R/R after ≥2 tyrosine kinase inhibitor therapies. Patients with priorblinatumomab were also included. Patients with chronic myelogenousleukemia lymphoid blast crisis or clinically significant infections werenot eligible. Patients with Burkitt leukemia/lymphoma were also noteligible.

For B cell NHL, key enrollment criteria included age <18 years, weight≥10 kg, histologically confirmed diffuse large B cell lymphoma nototherwise specified (DLBCL NOS), primary mediastinal large B celllymphoma, Burkitt lymphoma (BL), Burkitt-like lymphoma or unclassified Bcell lymphomas intermediate between DLBCL and BL, with ≥1 measurablelesion. For NHL, disease must have been primary refractory, R/R after ≥2lines of systemic therapy, or R/R after autologous or allogeneic stemcell transplantation ≥100 days prior to enrollment. Patients must havebeen off immunosuppressive medications for ≥4 weeks. The Lansky (age <16years) or Karnofsky (age ≥16 years) performance status was PS ≥80,weight ≥6 kg. Patients with prior blinatumomab were also included.Patients must have received adequate prior therapy, at a minimumanti-CD20 mAb and anthracycline-containing chemotherapy and have one ormore measurable lesions. Patients with acute graft-versus-host diseaseor chronic graft-versus-host disease requiring treatment within 4 weeksof enrollment were not eligible. Patients with prior CAR T cell therapyor other genetically modified T cell therapy were excluded, thoughpatients who received KTE-X19 in this study were eligible forre-treatment. Patients with cardiac lymphoma involvement or who requiredurgent therapy due to tumor mass effects were also excluded. Additionalexclusion to the ALL and NHL cohorts included: patients with clinicallysignificant infection; patients with acute or chronic GVHD requiringtreatment within 4 weeks of enrollment Alemtuzumab (or other anti-CD52antibody) within past 6 months, clofarabine or cladribine within past 3months, PEG-asparaginase within past 3 weeks, or donor leukocyteinfusion (DLI) within past 28 days.

Patients with CNS involvement and certain abnormalities were excluded.Patients with central nervous system-1 disease (no detectablelymphoblasts in cerebrospinal fluid), central nervous system-2 disease(detectable disease, but white blood cell count <5/μL in cerebrospinalfluid) with presence of lymphoblasts and with neurologic symptoms andwithout clinically evident neurologic changes who had prior blinatumomabtreatment may have been included in the ALL and NHL cohorts. Patientswith presence of lymphoblasts and with neurologic symptoms, centralnervous system-3 disease (WBC ≥5/μL in CSF) disease with presence oflymphoblasts with or without neurologic symptoms, patients with any CNStumor mass by imaging and/or parameningeal mass, history or presence ofany CNS disorder such as cerebrovascular ischemia/hemorrhage, dementia,cerebellar disease, or any autoimmune disease with CNS involvement,posterior reversible encephalopathy syndrome, or cerebral edema withstructural defects, history of stroke or transient ischemic attackwithin past 12 months, and seizure disorder requiring activeanti-convulsive medication were excluded. Patients with priorCD19-directed therapy, except for blinatumomab, were excluded.

Patients received conditioning chemotherapy with fludarabine 25 mg/m² onDays −4, −3, and −2 and cyclophosphamide 900 mg/m² on Day −2 followed bya single infusion of anti-CD19 CAR T cells at a target dose of 1×10⁶anti-CD19 CAR T cells/kg on Day 0. For ALL, the primary Phase 2objective was to evaluate anti-CD19 CAR T cell efficacy as assessed byoverall CR rate (CR and CR with incomplete hematologic recovery). ForNHL, the primary Phase 2 objective was to evaluate anti-CD19 CAR T celltherapy efficacy by objective response rate (CR+partial response).Secondary Phase 2 objectives for ALL and NHL cohorts included safety andtolerability, additional efficacy endpoints, and changes inpatient-reported outcome scores.

The CAR T cell treatment used in this study was described in priorexamples, such as Example 5 (also known as KTE-X19), which is anautologous anti-CD19 CAR T cell therapy for the treatment of R/R mantlecell lymphoma and other R/R hematologic malignancies. PBMCs from anapheresis product are enriched for T cells by CD4+/CD8+ positiveselection which results in removal of malignant cells. The resulting Tcells are activated with anti-CD3/anti-CD28 antibodies in the presenceof IL-2, retrovirally transduced to introduce the anti-CAR geneconstruct (FMC63-28Z CAR) and expanded to the desired dose. The expandedT cells may be frozen for transport and shipped back to the patient forinfusion. Axicabtagene ciloleucel is made by a different method asdescribed in, for example, Park J. H. et al. N Engl J Med. 2018;378(5):449-459; and Lee D. W. et al. Lancet. 2015; 385(9967):517-528. Inadult patients with R/R B-ALL, KTE-X19 treatment improved CR rate, CRirate, or safety profile in the Phase 1. Shah B D, et al. J Clin Oncol.2019; 37(suppl, abstr):7006.

During the DLT assessment in Phase 1, the starting dose was 2×10⁶anti-CD19 CAR T cell/kg. DLT was defined as Grade 3 nonhematologic AEslasting >7 days and Grade 4 nonhematologic AEs regardless of duration,with protocol-specified exceptions, or Grade 4 hematologic AEslasting >30 days. The dose of 1×10⁶ CAR-T cells in 68-mL volume or in40-mL volume was also examined. Patients receiving the 40-mL, 1×10⁶cohort received modified AE management. Based on available data, 1×10⁶cells/kg in 40-mL was used in Phase 2. Results of Phase 1 study showed94% of MRD-negativity and 73% of CR+Cri were observed in pediatric andadolescent patients with R/R B-ALL. Results also showed a manageable AEprofile consistent with known toxicities, and lower incidence andseverity of NEs with optimized dose formulation and revised safetymanagement. Wayne A S, et al. Pediatr Blood Cancer. 2019; 66(suppl):524.

In Phase 2, patients were screened and subject to leukapheresis followedby conditioning chemotherapy starting at Day −4. Bridging therapy mayhave been administered after leukapheresis at the investigator'sdiscretion and had to be completed ≥7 days or 5 half-lives beforeconditioning chemotherapy. KTE-X19 was infused at Day 0. The firstdisease assessment occurred at Day 28. Post-treatment assessment ofsafety and efficacy occurred on Week 2, Week 4, Month 2, and Month 3.Patients are followed up every 3 months through Month 18 and every 6months between Months 24 and 60. Beginning with year 6, patients returnonce annually for up to 15 years. A total of 50 patients with R/R ALLand 16 patients with R/R NHL were enrolled with the 40-mL formulation of1×10⁶ KTE-X19 cells/kg. The patients in Phase 2 of the current studyincluded also an NHL cohort and broadened enrollment criteria for R/RB-ALL to include patients with early first relapse, which was associatedwith poorer outcomes, as well as patients with MRD-positive disease.Primary objective was the efficacy as assessed by overall CR rate (CRand Cri) for ALL and by ORR (CR+PR) for NHL. Secondary objectivesincluded assessment of safety, tolerability, DOR, OS, relapse-freesurvival (RFS)/progression-free survival (PFS), and patient reportedoutcomes (PROs). For ALL, additional secondary objectives includedassessment of MRD-negative rate and allo-SCT rate. For the overall CRrate (ALL cohort only), incidence and exact 2-sided 95% CIs will bedetermined. It will be compared with a response rate of 35% at a 1-sidedα-level of 0.025 using an exact binomial test. For the MRD-negative rate(ALL cohort only), incidence and exact 2-sided 95% CIs will bedetermined. If statistical testing of the overall CR rate issignificant, MRD-negative rate will be compared to a rate of 30% at a1-sided α-level of 0.025 using an exact binomial test. For DOR and OS,Kaplan-Meier estimates and 2-sided 95% CIs will be determined. For theAlloSCT rate (ALL cohort only), incidence in mITT set and exact 2-sided95% CIs will be determined. In terms of safety, incidence rates of AEsincluding all, serious, fatal, CTCAE version 4.03 Grade ≥3, andtreatment-related AEs with onset on or after the date of infusion willbe determined. No specific hypothesis will be tested for the NHL cohort.With the planned sample size in this cohort, assuming an observed ORR of63% (10/16 patients), 69% (11/16), 75% (12/16), and 81% (13/16), thelower bound of the 95% exact CI for the estimated ORR will be 35%, 41%,48%, and 54%, respectively.

Example 9

This Example reports on the phase 1 results for ZUMA-3(ClinicalTrials.gov Identifier: NCT02614066), a phase 1/2 studyevaluating an autologous anti-CD19 chimeric antigen receptor (CAR)T-cell therapy that includes a CD3ζ and CD28 co-stimulatory domain andis prepared as described in the previous Examples (CD4+/CD8+enrichment/removal of malignant cells), in adults withrelapsed/refractory (R/R) B cell ALL. This protocol for preparation ofanti-CD19 CAR T cells with cancer cell removal reduces the likelihood ofactivation and exhaustion of anti-CD19 CAR T cells during ex vivomanufacturing. The presence of leukemic blasts in peripheral blood maylimit the number of T cells available for the manufacture CAR T-cellproducts, potentially leading to manufacturing failure. Sabatino M. etal. Blood. 2016; 128(22):1227. The anti-CD19 CAR T cell product used inthis study has been described in Wang M. et al. N Engl J Med. 2020;382(14):1331-1342 for use in MCL. It is different from that used inSabatino M. et al. Blood. 2016; 128(22):1227, Park J. H. et al. N Engl JMed. 2018; 378(5):449-459; and Lee D. W. et al. Lancet. 2015;385(9967):517-528. This anti-CD19 CAR T cell product has differentproduct characteristics in terms of T cell phenotype than that made bypreviously-described methods. This anti-CD19 CAR was also referred to asKTE-X19 in this example and elsewhere in the application.

Following fludarabine/cyclophosphamide lymphodepletion, patientsreceived anti-CD19 CAR T cells at 2, 1, or 0.5×10⁶ cells/kg. Rate ofdose-limiting toxicities (DLTs) within 28 days following CAR T cellinfusion was the primary endpoint. Anti-CD19 CAR T cells weremanufactured for 54 enrolled patients and administered to 45 (median age46 years [range, 18-77]). No DLTs occurred in the DLT-evaluable cohort.Grade ≥3 cytokine release syndrome (CRS) and neurologic events (NE)occurred in 31% and 38% of patients, respectively. To optimize thebenefit-risk ratio, revised adverse event (AE) management for CRS and NE(earlier steroid use for NE and tocilizumab only for CRS) was evaluatedat 1×10⁶ cells/kg anti-CD19 CAR T cells. In the 9 patients treated underrevised AE management, 33% had grade 3 CRS and 11% had grade 3 NE, withno grade 4/5 NE. The overall complete remission rate correlated with CART cell expansion and was 83% in patients treated with 1×10⁶ cells/kg and69% in all patients. Minimal residual disease was undetectable in allresponding patients. At 22.1 months (range, 7.1-36.1) median follow-up,the median DOR was 17.6 months (range, 5.8-17.6) in patients treatedwith 1×10⁶ cells/kg and 14.5 months (range, 5.8-18.1) in all patients.Anti-CD19 CAR T cell treatment provided a high response rate andtolerable safety in adults with R/R B-ALL. Phase 2 proceeded at 1×10⁶cells/kg with revised AE management.

Patients

Eligible patients were ≥18 years of age with R/R B cell ALL, defined asrefractory to first-line therapy (i.e., primary refractory), relapse ≤12months after first remission, relapsed or refractory after ≥2 priorlines of systemic therapy, or relapsed after allogeneic stem celltransplant (SCT). Patients were required to have ≥5% bone marrow blasts,an Eastern Cooperative Oncology Group performance status of 0 or 1, andadequate renal, hepatic, and cardiac function. The first six patientsenrolled were required to have ≥25% blasts in bone marrow. For patientswho received prior blinatumomab, leukemic blasts with CD19 expression≥90% was required. Patients with Philadelphia chromosome-positive (Ph+)disease, concomitant extramedullary disease, central nervous system(CNS)-2 disease (cerebrospinal fluid [CSF] blast cells with <5 whiteblood cells/mm³) without neurological changes and patients with Downsyndrome were eligible. CNS-3 disease (CSF blast cells with ≥5 whiteblood cells/mm³) independent of neurologic changes and a history of CNSdisorder were exclusions.

Additional eligibility criteria included: Subjects with Philadelphiachromosome (Ph)+ disease were eligible if they had disease intolerant totyrosine kinase inhibitor (TKI) therapy, or if they hadrelapsed/refractory disease despite treatment with ≥2 different TKIs;Absolute neutrophil count ≥500/μL unless in the opinion of theinvestigator cytopenia is due to underlying leukemia and is potentiallyreversible with leukemia therapy; Platelet count ≥50,000/μL unless inthe opinion of the investigator cytopenia is due to underlying leukemiaand is potentially reversible with leukemia therapy; Absolute lymphocytecount ≥100/μL; Adequate renal, hepatic, pulmonary and cardiac functionwere defined [Creatinine clearance (as estimated by Cockcroft Gault)≥60cc/min; Serum alanine aminotransferase/aspartate aminotransferase≤2.5×upper limit of normal; Total bilirubin ≤1.5 mg/dL, except insubjects with Gilbert's syndrome; Left ventricular ejection fraction≥50%, no evidence of pericardial effusion as determined by anechocardiogram, no New York Heart Association class III or class IVfunctional classification, and no clinically significant arrhythmias; Noclinically significant pleural effusion; Baseline oxygen saturation >92%on room air]; Females of childbearing potential must have had a negativeserum or urine pregnancy test; Females of childbearing potential musthave had a negative serum or urine pregnancy test.

Additional exclusion criteria included: Diagnosis of Burkitt'sleukemia/lymphoma according to World Health Organization classificationor chronic myelogenous leukemia lymphoid blast crisis; History ofmalignancy other than non-melanoma skin cancer or carcinoma in situ (eg,cervix, bladder, breast) unless disease-free for ≥3 years; History ofsevere hypersensitivity reaction to aminoglycosides or any of the agentsused in this study; Central nervous system (CNS) abnormalities [Presenceof CNS-3 disease defined as detectable cerebrospinal blast cells in asample of cerebrospinal fluid (CSF) with ≥5 white blood cells (WBCs) permm³ with or without neurological changes, and; Presence of CNS-2 diseasedefined as detectable cerebrospinal blast cells in a sample of CSF with<5 WBCs per mm³ with neurological changes. Note: Subjects with CNS-1 (nodetectable leukemia in the CSF) and those with CNS-2 without clinicallyevident neurological changes are eligible to participate in the study;History or presence of any CNS disorder such as a seizure disorder,cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, anyautoimmune disease with CNS involvement, posterior reversibleencephalopathy syndrome, or cerebral edema]; History of severehypersensitivity reaction to aminoglycosides or any of the agents usedin this study; History of concomitant genetic syndrome associated withbone marrow failure; History of clinically significant cardiac diseasewithin 12 months of enrollment; History of symptomatic deep veinthrombosis or pulmonary embolism within 6 months of enrollment; Primaryimmunodeficiency; Known infection with HIV, hepatitis B, or hepatitis Cvirus. A history of hepatitis B or hepatitis C is permitted if the viralload is undetectable per quantitative polymerase chain reaction and/ornucleic acid testing; Simple urinary tract infection and uncomplicatedbacterial pharyngitis are permitted if responding to active treatmentand after consultation with the Kite Medical Monitor; Acutegraft-vs-host disease (GVHD) grade II-IV by Glucksberg criteria orseverity B-D by International Bone Marrow Transplant Registry index;acute or chronic GVHD requiring systemic treatment within 4 weeks priorto enrollment; Prior medication [Salvage systemic therapy (includingchemotherapy, TKIs for Ph+ disease, and blinatumomab)≤1 blinatumomab;History of Common Terminology Criteria for Adverse Events grade 4neurologic event or grade 4 cytokine release syndrome with priorCD19-directed therapy; Treatment with alemtuzumab ≤6 months prior toenrollment, clofarabine or cladribine ≤3 months prior to enrollment orPEG-asparaginase ≤3 months prior to enrollment; Donor lymphocyteinfusion ≤4 weeks prior to enrollment; Treatment with any drug for GVHDand any immunosuppressive antibody ≤4 weeks prior to enrollment; Atleast 3 half-lives must have elapsed from any prior systemicinhibitory/stimulatory immune checkpoint molecular therapy prior toenrollment; Corticosteroid therapy at a pharmacologic dose (>5 mg/day ofprednisone or equivalent doses of other corticosteroids) and otherimmunosuppressive drugs must be avoided for 1 week prior to enrollment];Presence of any indwelling line or drain. Ommaya reservoirs anddedicated central venous access catheters are permitted; Live vaccine ≤4weeks prior to enrollment; Women of childbearing potential who arepregnant or breastfeeding because of the potentially dangerous effectsof the preparative chemotherapy on the fetus or infant; Subjects of bothgenders of childbearing potential who are not willing to practice birthcontrol from the time of consent through 6 months after the completionof anti-CD19 CAR T cell therapy; Subjects who, in the investigator'sjudgment, are unlikely to complete all protocol-required study visits orprocedures or comply with the study requirements for participation[History of autoimmune disease resulting in end organ injury orrequiring systemic immunosuppression or systemic disease modifyingagents within the last 2 years].

Study Design and Treatment

The phase 1 objective was to evaluate the safety of anti-CD19 CAR T celltreatment and determine the optimal phase 2 dose based on the incidenceof dose-limiting toxicities (DLTs) and overall safety profile. DLTs weredefined as anti-CD19 CAR T cell-related adverse events (AEs) occurringwithin the first 28 days following anti-CD19 CAR T cell infusion,including grade 3 non-hematologic AEs lasting >7 days, grade 4non-hematologic AEs regardless of duration except for prespecifiedexpected events (e.g., tumor lysis syndrome), and grade 4 hematologicAEs lasting >30 days, except lymphopenia (Table 15).

TABLE 15 Dose-limiting toxicities DLTs were defined as the followinganti-CD19 CAR T cells-related events with onset within the first 28 daysfollowing anti-CD19 CAR T cells infusion: Grade 4 hematologic toxicitylasting more than 30 days (except lymphopenia) if not attributable tounderlying disease All anti-CD19 CAR T cell-related grade 3non-hematologic toxicities lasting for > 7 days and all anti-CD19 CAR Tcell-related grade 4 non-hematologic toxicities regardless of durationwere considered DLTs, with the exception of the following:Aphasia/dysphasia or confusion/cognitive disturbance which resolved toat least grade 1 or baseline within 2 weeks and to at least baselinewithin 4 weeks Fever grade 3 or 4 Immediate hypersensitivity reactionsoccurring within 2 hours of anti-CD19 CAR T cells infusion (related toanti-CD19 CAR T cells infusion) that were reversible to a grade 2 orless within 24 hours of anti-CD19 CAR T cell infusion with standardtherapy Renal toxicity which required dialysis for ≤ 7 days Intubationfor airway protection if ≤ 7 days TLS including associatedmanifestations attributable to TLS (eg, electrolyte abnormalities, renalfunction, hyperuricemia) Grade 3 transaminase, alkaline phosphatase,bilirubin or other liver function test elevation, provided there wasresolution to ≤ grade 2 within 14 days Grade 4 transient serum hepaticenzyme abnormalities provided there was resolution to ≤ grade 3 within <72 hours Hypogammaglobulinemia grade 3 or 4 Grade 3 nausea and/oranorexia Adverse events attributed to CRS were mapped to the overall CRSgrading assessment for the determination of DLT All occurrences of grade3 CRS of duration > 7 days and all occurrences of grade 4 CRS wereconsidered DLTs, other than occurrences of CRS due to the exceptionslisted above CRS, cytokine release syndrome; DLT, dose-limitingtoxicity; TLS, tumor lysis syndrome

Initial patients were enrolled at a starting dose of 2×10⁶ CAR Tcells/kg (FIG. 3 ). Based on the overall safety profile, subsequentpatients received 2×10⁶, 1×10⁶, or 0.5×10⁶ CAR T cells/kg. At 0.5×10⁶CAR T cells/kg. Two formulations were explored for patients receivingthe lower dose 0.5×10⁶ CAR T cells/kg, one with a total volume of 40 mLand the other with a volume of 68 mL. The 40-mL formulation was intendedto maintain cell density and cell viability during the freezing/thawingprocess).

To mitigate the risk of cytokine release syndrome (CRS) and neurologicevents (NE), AE management guidelines were revised to limit tocilizumabto the treatment of CRS (and not isolated neurotoxicity), and toinitiate corticosteroid treatment at the onset of grade 2 rather thangrade 3 NE (Table 16).

TABLE 16 Original and revised neurotoxicity management guidelines NEGrade Original Management Guidelines Revised Management Guidelines Grade1 Supportive care Supportive care Neurological examination and Closelymonitor neurologic status additional work-up as clinically Considerprophylactic antiepileptic indicated Grade 2 Supportive Care andEvaluation Supportive Care and Evaluation Neurological examination,brain MRI, Continuous cardiac telemetry and and evaluation of CSF;consider EEG as pulse oximetry as indicated clinically indicated Serialneurological examinations to Consider prophylactic antiepileptic includefundoscopy and Glasgow Coma Score, brain MRI, evaluation of CSF, EEG;consider neurology consult Administer antiepileptics for patients withseizures Tocilizumab Tocilizumab Consider tocilizumab 8 mg/kg IV over 1For patients with concurrent CRS, hour (not to exceed 800 mg) forpatients administer tocilizumab 8 mg/kg IV with comorbid conditions (eg,grade ≥ 2 over 1 hour (not to exceed 800 mg); CRS) repeat every 4-6hours as needed if not responsive to IV fluids or increasingsupplemental oxygen, for a maximum of 3 doses in 24 hours Discontinuetocilizumab if patient improves Corticosteroids Corticosteroids N/A Forpatients without concurrent CRS, administer dexamethasone 10 mg IV every6 hours For patients with concurrent CRS, if no improvement within 24hours after starting tocilizumab, administer dexamethasone 10 mg IVevery 6 hours Taper corticosteroids if patient improves Grade 3Supportive Care and Evaluation Supportive Care and Evaluation Per grade2 Manage in monitored care or ICU Monitor with continuous cardiactelemetry and pulse oximetry Tocilizumab Tocilizumab Considertocilizumab 8 mg/kg IV over 1 Per grade 2 hour (not to exceed 800 mg);repeat Discontinue tocilizumab if patient every 4-6 hours if symptomshave not improves stabilized or improved Corticosteroids CorticosteroidsConsider corticosteroids (eg, Administer dexamethasone 10 mg IVdexamethasone 10 mg IV every 6 hours every 6 hours or methylprednisolonel mg/kg BID) Taper corticosteroids if patient for worsening symptomsdespite improves tocilizumab Grade 4 Supportive Care and EvaluationSupportive Care and Evaluation Per grade 2 Per grade 3 Monitor withcontinuous cardiac Mechanical ventilation may be telemetry and pulseoximetry required Administer immunosuppresants if patient does notimprove Tocilizumab Tocilizumab Administer tocilizumab per grade 3 ifPer grade 2 not previously administered Corticosteroids CorticosteroidsAdminister corticosteroids (eg, Administer high-dose corticosteroidsmethylprednisolone ig/d × 3 days, (eg, methylprednisone ig/d × 3 days)followed by 250 mg BID × 2 days, then Taper corticosteroids if patient125 mg BID × 2 days, then 60 mg improves BID × 2 days)

Revised AE management guidelines were implemented in an additionalcohort of patients treated with 1×10⁶ CAR T cells/kg. A safety reviewteam (SRT) reviewed safety and efficacy data on an ongoing basis andmade recommendations regarding further phase 1 enrollment and therecommended phase 2 dose (RP2D) at milestones defined in the protocoland SRT charter.

Patients underwent leukapheresis at enrollment to obtain a target of5-10×10⁹ mononuclear cells for anti-CD19 CART cells manufacture.Predefined bridging chemotherapy (Table 17) was recommended followingleukapheresis, particularly for patients with high disease burden atbaseline (>25% leukemic blasts in bone marrow or ≥1,000 blasts/mm³ inperipheral circulation by local review).

TABLE 17 Bridging chemotherapy Predefined Bridging Chemotherapy RegimensAttenuated VAD Vincristine non-liposomal (1-2 mg IV weekly) or liposomal(2.25 mg/m² IV weekly), and dexamethasone 20-40 mg IV or PO daily × 3-4days per week. Optional doxorubicin 50 mg/m² IV × 1 (first week only)Mercaptopurine (6- 50-75 mg/m²/day by mouth (administer at bedtime on anempty MP) stomach to improve absorption) Hydroxyurea Doses titratedbetween 15-50 mg/kg/day (rounded to the nearest 500 mg capsule and givenas a single daily oral dose on a continuous basis) DOMP Dexamethasone 6mg/m²/day PO (or IV) divided BID days 1-5, vincristine 1.5 mg/m²(maximum dose 2 mg) IV on day 1, methotrexate 20 mg/m² PO weekly, 6-MP50-75 mg/m²/day PO daily Attenuated Fludarabine 30 mg/m² IV days 1-2,cytarabine 2 g/m² IV days 1-2, FLAG/FLAG-IDA G-CSF 5 μg/kg SC or IVstarts on day 3 and can continue until day before the start ofconditioning chemotherapy. With or without idarubicin 6 mg/m² IV days1-2 Mini-hyper CVAD Course A: Cyclophosphamide 150 mg/m² every 12 h × 3days, (courses A and/or B) dexamethasone 20 mg/d IV or PO daily days 1-4and 11-14, vincristine 2 mg IV × 1 Course B: methotrexate 250 mg/m² IVover 24 hours on day 1, cytarabine 0.5 g/m² IV every 12 hours × 4 doseson days 2 and 3 BID, twice daily; CVAD, cyclophosphamide, vincristine,doxorubicin, and dexamethasone; DOMP, dexamethasone, 6-mercaptopurine,methotrexate, and vincristine; FLAG, fludarabine, high-dose cytarabine,and G-CSF; G-CSF, granulocyte-colony stimulating factor; IDA,idarubicin; IV, intravenous; MP, 6-mercaptopurine; PO, oral; SC,subcutaneous; VAD, vincristine, doxorubicin, and dexamethasone.

After ≥7 days or 5 half-lives (if shorter) washout from bridgingchemotherapy, patients received a lymphodepleting regimen of fludarabineintravenous (IV) 25 mg/m²/day on days −4, −3, and −2, andcyclophosphamide IV 900 mg/m²/day on day −2. On day 0, a single infusionof anti-CD19 CAR T cells was administered.

Outcomes and Assessments

The primary phase 1 endpoint was the incidence of DLTs in DLT-evaluablepatients. Secondary endpoints included safety, investigator-assessedoverall remission rate (CR+CR with incomplete hematologic recovery[CRi]), duration of remission (DOR), relapse-free survival, OS, and rateof undetectable minimal residual disease (MRD) in bone marrow. Levels ofCAR T cells and cytokines in blood were exploratory endpoints. AEsincluding symptoms of CRS and NE were graded per the Common TerminologyCriteria for AEs version 4.03. CRS was graded per the criteria of Lee,et al. Blood. 2014; 124(2):188-195. For patients with extramedullarydisease, response was assessed per the response criteria forextramedullary and CNS disease in the revised International WorkingGroup Criteria for malignant lymphoma. Cheson B D et al. J Clin Oncol.2007; 25(5):579-586. Undetectable MRD, defined as <1 leukemia cell per10,000 viable cells, was centrally assessed using flow cytometry(NeoGenomics, Fort Myers, Fla.). Borowitz M J et al Blood. 2015;126(8):964-971; Bruggemann M. et al. Blood Adv. 2017; 1(25):2456-2466;and Gupta S. et al. Leukemia. 2018; 32(6):1370-1379.

Hospitalization for ≥7 days post-infusion was required. Patients wereevaluated at days 14 and 28 and months 2 and 3 by physical examinations,vital sign measurements, and neurological and laboratory assessments.Bone marrow evaluations and response assessments were conducted at days7-14 (optional) and 28 and months 2 and 3. For patients who underwentSCT post-anti-CD19 CAR T cell infusion, bone marrow evaluation was notrequired during the first 100 days post-SCT. Collection and analysis ofCSF was required to confirm CR for patients with baseline CNS-2 disease.Patients completing month 3 post-treatment assessments were followed forsurvival and disease status every 3 months through month 18, every 6months during months 24-60, and annually for up to 15 years. Patientsachieving CR could receive a second infusion of anti-CD19 CART cells ifprogressing following >3 months of remission, provided CD19 expressionwas retained and neutralizing antibodies against the CAR were notsuspected.

Biomarker analyses were performed on blood and serum samples to evaluatepredictive pharmacokinetics and pharmacodynamic markers for anti-CD19CAR T cells. As previously described, droplet digital polymerase chainreaction was used to measure the presence, expansion, and persistence oftransduced CD19 CAR+ T cells in blood Locke Fla. et al. Mol Ther. 2017;25(1):285-295. Serum was assessed for cytokines, chemokines, immuneeffector molecules, and markers of macrophage-activating syndrome usingpreviously reported methods. Locke Fla. et al. Mol Ther. 2017;25(1):285-295.

Statistical Analysis

The DLT-evaluable cohort included the first 3 patients treated at the2×10⁶ dose level. Safety and efficacy analyses included all patientstreated with any dose of anti-CD19 CAR T cells. Kaplan-Meier estimatesand 2-sided 95% confidence intervals were generated for time-to-eventendpoints. DOR was defined as time from CR to relapse or death withoutdocumented relapse. The DOR for patients who underwent allogeneic SCTwhile in remission was censored at the date of transplant. OS wasdefined as time from anti-CD19 CAR T cell infusion to date of death fromany cause. Data are presented as of Apr. 1, 2019. All statisticalanalyses were done in SAS (version 9.4).

Results

Patients

Between Mar. 9, 2016 and Jul. 12, 2018, 54 patients were enrolled andunderwent leukapheresis in phase 1 (FIG. 4 ). The anti-CD19 CAR T cellproduct was successfully manufactured for all 54 patients; 1 patientrequired 2 leukapheresis procedures and 1 patient required 3 forprocedures for product manufacturing. The median time from leukapheresisto delivery of anti-CD19 CAR T cells to the study site was 15 days. Fivepatients discontinued prior to lymphodepletion because of AEs (n=3; FIG.4 ), withdrawal of consent (n=1), or ineligibility after leukapheresis(n=1). Four additional patients discontinued following lymphodepletion.Three received no anti-CD19 CAR T cells due to grade 4 sepsis (n=1),initiation of new therapy (n=1), and death from grade 5 sepsis (n=1).One patient discontinued prior to infusion due to deep vein thrombosis(an exclusion criterion) but received anti-CD19 CAR T cells undercompassionate use. Forty-five of 54 patients (83%) received anti-CD19CAR T cells at these dose levels: 2×10⁶ (n=6), 1×10⁶ (n=23), or 0.5×10⁶CAR T cells/kg (n=16). Nine of 23 patients in the 1×10⁶ CAR T cells/kgcohort were treated under revised AE management guidelines requiringearlier use of steroids for NE and reserving tocilizumab only fortreating CRS. Forty-four patients received their target dose ofanti-CD19 CAR T cells; 1 patient enrolled to receive 1×10⁶ anti-CD19 CART cells/kg and revised AE management was treated with 0.5×10⁶ cells/kg,but was included in the analysis at the 1×10⁶ dose level.

The median age of all treated patients was 46 years (range, 18-77), and67% received ≥3 prior lines of therapy (Table 18). Prior to enrollment,16 patients (40%) were primary refractory, 13 (29%) relapsed after SCT,and 21 (47%) received prior blinatumomab. Blinatumomab was the lasttherapy used before study entry in 8 patients (18%), only 1 of whomachieved a response (CR) to blinatumomab.

Table 18 Patient baseline characteristics Baseline Characteristics N =45 Age, median (range), y 46 (18-77) Male, n (%) 22 (49) ECOGperformance status score, n (%)  0 15 (33)  1 29 (64) Missing 1 (2)Philadelphia chromosome-positive, n (%) 8 (18) Extramedullary disease, n(%) 4 (9) CNS disease at screening, n (%) CNS-1 42 (93) CNS-2 3 (7)Prior regimens, n (%)  1 6 (13)  2 9 (20) ≥3 30 (67) Prior blinatumomab,n (%) 21 (47) Prior inotuzumab ozogamicin, n (%) 6 (13) Refractory, n(%) Primary refractory 16 (36) First relapse with remission ≤12 months 2(4) Relapsed or refractory post-allogeneic SCT 13 (29) BM blasts atscreening, median (range), % 61 (5-100) BM blasts at preconditioningafter bridging, 70 (0-97) median (range), % BM, bone marrow; CNS,central nervous system; ECOG, Eastern Cooperative Oncology Group; SCT,stem cell transplant

Safety

No DLTs were observed among the DLT-evaluable set (n=3). Ninety-eightpercent of patients experienced grade ≥3 AEs (Table 19). The most commonany-grade AEs were pyrexia (89%), hypotension (69%), diarrhea (42%), andchills (42%). Common grade ≥3 AEs (≥20% of patients) were pyrexia (42%),hypotension (40%), platelet count decreased (33%), anemia (31%),hypophosphatemia (31%), hypoxia (24%), encephalopathy (22%), febrileneutropenia (22%), and neutrophil count decreased (22%). Serious AEs ofany grade occurred in 84% of patients.

TABLE 19 Adverse events 2 × 10⁶ 1 × 10⁶ 0.5 × 10⁶ All Patients n (%)* (n= 6) (n = 23) (n = 16) (N = 45) Any adverse event 6 (100) 6 (100) 23(100) 23 (100) 16 (100) 15 (94) 45 (100) 44 (98) Pyrexia 6 (100) 3 (50)22 (96) 11 (48) 12 (75) 5 (31) 40 (89) 19 (42) Hypotension 5 (83) 3 (50)17 (74) 11 (48) 9 (56) 4 (25) 31 (69) 18 (40) Chills 3 (50) 0 13 (57) 03 (19) 0 19 (42) 0 (0) Diarrhea 3 (50) 0 10 (43) 1 (4) 6 (38) 0 19 (42)1 (2) Headache 1 (17) 0 10 (43) 1 (4) 7 (44) 1 (6) 18 (40) 2 (4) Anemia4 (67) 4 (67) 10 (43) 8 (35) 3 (19) 2 (13) 17 (38) 14 (31)Encephalopathy 4 (67) 2 (33) 11 (48) 6 (26) 2 (13) 2 (13) 17 (38) 10(22) Hypophosphatemia 2 (33) 1 (17) 12 (52) 10 (43) 3 (19) 3 (19) 17(38) 14 (31) Nausea 1 (17) 0 13 (57) 1 (4) 3 (19) 0 17 (38) 1 (2)Confusional state 2 (33) 1 (17) 9 (39) 1 (4) 5 (31) 2 (13) 16 (36) 4 (9)Hypoxia 2 (33) 1 (17) 8 (35) 6 (26) 6 (38) 4 (25) 16 (36) 11 (24)Platelet count 3 (50) 3 (50) 8 (35) 8 (35) 5 (31) 4 (25) 16 (36) 15 (33)decreased Constipation 2 (33) 0 10 (43) 0 2 (13) 0 14 (31) 0 (0) Fatigue1 (17) 0 7 (30) 1 (4) 6 (38) 0 14 (31) 1 (2) Sinus tachycardia 2 (33) 010 (43) 1 (4) 2 (13) 0 14 (31) 1 (2) Hypokalemia 1 (17) 0 11 (48) 0 1(6) 0 13 (29) 0 (0) Tachycardia 1 (17) 1 (17) 6 (26) 1 (4) 6 (38) 0 13(29) 2 (4) Tremor 1 (17) 0 8 (35) 0 4 (25) 0 13 (29) 0 (0) Decreasedappetite 0 0 9 (39) 2 (9) 3 (19) 0 12 (27) 2 (4) Hyperglycemia 1 (17) 06 (26) 0 5 (31) 1 (6) 12 (27) 1 (2) Hypomagnesemia 2 (33) 0 8 (35) 0 2(13) 0 12 (27) 0 (0) Hyponatremia 3 (50) 2 (33) 7 (30) 3 (13) 2 (13) 012 (27) 5 (11) Edema peripheral 1 (17) 0 7 (30) 1 (4) 4 (25) 0 12 (27) 1(2) *Table includes adverse events of any grade occurring in ≥25% of allpatients

CRS was reported in 42 patients (93%); 14 patients (31%) experiencedgrade ≥3 CRS (Table 19). Common grade ≥3 symptoms of CRS were pyrexia(45%), hypotension (36%), and hypoxia (17%). Vasopressors were used forthe treatment of CRS in 12 patients (27%). The median time to CRS onsetpost-infusion was 2 days (range, 1-12); the median durations of anygrade and grade ≥3 CRS were 9 and 4.5 days, respectively. CRS-associatedevents resolved in all but the 2 patients who experienced grade 5anti-CD19 CAR T cell-related AEs. One patient treated with 2×10⁶ CAR Tcells/kg had multiorgan failure secondary to CRS (day 6). One patienttreated with 0.5×10⁶ cells/kg developed cerebrovascular accident(stroke) in the context of CRS and NE (day 7). No other anti-CD19 CAR Tcell-related grade 5 AEs were reported.

NE were reported in 35 patients (78%); grade ≥3 events occurred in 17patients (38%; Table 19). Grade ≥3 NE occurring in ≥5% of patients wereencephalopathy (22%), aphasia (16%), and confusional state (9%). Therewere no cases of cerebral edema and no grade 5 NE. The median time toonset of NE was 6 days (range, 1-31) after infusion; the mediandurations of any grade and grade ≥3 NE were 12 and 9 days, respectively.NE resolved in 31/35 patients (89%); 1 patient died from progressivedisease and 3 patients died from AEs considered unrelated to anti-CD19CART cells (sepsis [n=1], cerebrovascular accident [n=1], herpes simplexviremia [n=1]) prior to neurologic event resolution.

Fifty-three percent of all patients received tocilizumab, and 36% alsoreceived steroids for management of CRS; 31% and 44% receivedtocilizumab and steroids, respectively, for NE. Improved overall safetywas observed for the 9 patients treated under revised AE managementguidelines relative to the 14 patients treated at the same dose underthe original guidelines (Table 20). Four of 14 patients treated at 1×10⁶CAR T cells/kg under the original guidelines had grade 3 or 4 CRS. Withrevised AE management, 3/9 patients treated at 1×10⁶ CAR T cells/kg hadgrade 3 CRS, with no grade 4 CRS reported. These patients also had ashorter median duration of grade ≥3 CRS (4 vs. 7 days) than patientsreceiving 1×10⁶ CAR T cells/kg under original AE guidelines, and alonger time to onset of grade ≥3 symptoms (6 vs. 4.5 days,respectively). Notably, 9/14 patients in the 1×10⁶ CAR T cells/kg dosecohort managed with the original guidelines experienced grade 3/4 NE,compared to one grade 3 and no grade 4 events in patients receiving thesame dose under revised management guidelines (Table 20). Based on thereview of all available safety and efficacy data, the benefit/risk ratiowas considered most favorable at the dose of 1×10⁶ CAR T cells/kg,resulting in this dose being the RP2D. All phase 2 patients were beingtreated under revised AE management guidelines.

TABLE 20 Cytokine release syndrome and neurologic events including withrevised AE management guidelines 1 × 10⁶ 1 × 10⁶ Original AE Revised AE2 × 10⁶ Management Management 0.5 × 10⁶ n (%) (n = 6) (n = 14) (n = 9)(n = 16) Steroids For treatment of 1 (17) 5 (36) 5 (56) 5 (31) CRS Fortreatment of 3 (50) 7 (50) 5 (56) 5 (31) NE Tocilizumab For treatment of1 (17) 9 (64)  9 (100) 5 (31) CRS For treatment of 4 (67) 5 (36) 4 (44)1 (6)  NE Any Any Any Any Adverse event, n (%) grade Grade ≥3 gradeGrade ≥3 grade Grade ≥3 grade Grade ≥3 Cytokine release 6 (100) 3 (50)14 (100) 4 (29) 9 (100) 3 (33) 13 (81) 4 (25) syndrome Pyrexia 6 (100) 3(50) 12 (86) 5 (36) 9 (100) 6 (67) 10 (77) 5 (31) Hypotension 4 (67) 3(50) 11 (79) 6 (43) 6 (67) 3 (33) 8 (62) 3 (19) Sinus tachycardia 2 (33)0 6 (43) 0 4 (44) 1 (11) 2 (15) 0 Chills 1 (17) 0 5 (36) 0 4 (44) 0 2(15) 0 Tachycardia 1 (17) 1 (17) 4 (29) 1 (7) 2 (22) 0 4 (31) 0Tachypnea 0 0 4 (29) 1 (7) 0 0 0 0 Hypoxia 2 (33) 1 (17) 2 (14) 2 (14) 3(33) 2 (22) 3 (23) 2 (15) Nausea 0 0 2 (14) 0 0 0 0 0 Fatigue 0 0 1 (7)0 3 (33) 0 1 (8) 0 Headache 0 0 1 (7) 0 2 (22) 0 3 (23) 0 Hyponatremia 00 1 (7) 0 1 (11) 0 1 (8) 0 Any neurologic event 5 (83) 3 (50) 13 (93) 9(64) 7 (78) 1 (11) 10 (63) 4 (25) Confusional state 2 (33) 1 (17) 3 (21)0 6 (67) 1 (11) 5 (31) 2 (13) Tremor 1 (17) 0 4 (29) 0 4 (44) 0 4 (25) 0Aphasia 0 0 6 (43) 4 (29) 2 (22) 1 (11) 2 (13) 2 (13) Encephalopathy 4(67) 2 (33) 9 (64) 6 (43) 2 (22) 0 2 (13) 2 (13) Lethargy 0 0 1 (7) 0 2(22) 0 2 (13) 0 Mental status 0 0 0 0 2 (22) 0 0 0 changes Agitation 0 04 (29) 1 (7) 1 (11) 1 (11) 2 (13) 0 Dysarthria 0 0 1 (7) 1 (7) 1 (11) 00 0 Restlessness 0 0 1 (7) 1 (7) 1 (11) 1 (11) 0 0 Seizure 1 (17) 0 2(14) 2 (14) 1 (11) 0 1 (6) 0 Ataxia 0 0 1 (7) 0 1 (11) 0 0 0 AE, adverseevent; CRS, cytokine release syndrome; NE, neurologic events

Twenty-six treated patients (58%) died from causes that included diseaseprogression in 19 (42%) and AEs in 7 patients (16%), including the 2above-mentioned treatment-related deaths. The remaining 5 AE-relateddeaths occurred at a median 63 days (range, 48-579) after infusion ofanti-CD19 CAR T cells and were considered unrelated to anti-CD19 CAR Tcells. They included sepsis (n=2), cerebrovascular accident (n=1),herpes simplex viremia (n=1) and bacteremia (n=1).

Efficacy

All 45 treated patients were eligible for efficacy analysis. At a medianfollow-up of 22.1 months (range, 7.1-36.1), the overall remission rate(ORR) was 69%, with 51% of patients achieving CR and 18% CRi (Table 21).Among the 23 patients treated with 1×10⁶ CAR T cells/kg, the ORR was83%, with 14 achieving CR (61%) and 5 (22%) CRi. Six of 9 patients whoreceived revised AE management achieved CR/CRi (4 CR, 2 CRi). The mediantime to CR/CRi across dose levels was 30 days (range, 26-192), whichincluded 1 patient with blast-free hypoplastic/aplastic bone marrow(BFBM) at day 28 who did not meet CR criteria until month 6. ORR wasgenerally consistent across key covariates, including refractorypatients (56%), prior transplant (77%), prior blinatumomab (57%) orinotuzumab ozogamicin (50%), and Ph+ disease patients (100%) (FIG. 5 ).Undetectable bone marrow MRD was achieved at day 28 in 100% ofresponders, including the 31 patients with CR/CRi, 1 patient withpartial response, and 1 with BFBM. Residual disease assessment wasunavailable in 1 patient with BFBM. Two of 6 patients who underwent theoptional bone marrow assessment at day 7-14 had undetectable MRD; the 5patients with data available at day 30 had undetectable MRD.

TABLE 21 Response to anti-CD19 CAR T cells 2 × 10⁶ 1 × 10⁶ 0.5 × 10⁶Total Response Category, n (%) (n = 6) (n = 23) (n = 16) (N = 45)Complete remission 4 (67) 19 (83) 8 (50) 31 (69) Complete remission 3(50) 14 (61) 6 (38) 23 (51) Complete remission with 1 (17)  5 (22) 2(13)  8 (18) incomplete hematologic recovery Blast-freehypoplastic/aplastic 0 1 (4) 1 (6)  2 (4) bone marrow Partial remission0  1 (4)* 0 1 (2) No response 1 (17) 2 (9) 6 (3)   8 (18) Unknown or notevaluable  1 (17)^(†) 0 1 (6)^(‡) 2 (4) *Patient had extramedullarydisease at response assessment. ^(†)Patient died on day 6 due tomultiorgan failure secondary to CRS. ^(‡)Patient died on day 7 due tocerebrovascular accident (stroke) in the context of CRS and neurologicevents

The median DOR for the 31 patients achieving CR/CRi was 14.5 months (95%CI, 5.8-18.1; FIG. 6A), and 17.6 months (95% CI, 5.8-17.6) in patientstreated with 1×10⁶ CART cells/kg. Median DOR was similar regardless ofcensoring for SCT post-anti-CD19 CART cells (FIG. 6B). As of the datacutoff, 8 patients (26%) had ongoing CRs, including 2 who received0.5×10⁶ CAR T cells/kg and 6 who received 1×10⁶ CAR T cells/kg, with amedian follow-up of 6.3 months (range, 5.9-18.2). Six patients (2 CR and1 partial response treated with 1×10⁶ CAR T cells/kg; 3 CR treated with0.5×10⁶ cells/kg) underwent SCT at a median of 2.7 months (range,1.7-4.3) post-infusion. As of this analysis, 3 of these remained in CR(2 treated with 1×10⁶ CAR T cells/kg and 1 with 0.5×10⁶ cells/kg).Across all dose levels, the median duration of relapse-free survival was7.3 months (95% CI, 2.7-18.7) vs. 7.7 months (95% CI, 3.2-18.7) inpatients receiving 1×10⁶ CAR T cells/kg (FIG. 6C). The median OS was12.1 months (95% CI, 6.1-19.1) across all dose levels and 16.1 months(95% CI, 10.2—not estimable) with 1×10⁶ CAR T cells/kg (FIG. 6D).

As of the data cutoff, 1 patient (2%) withdrew consent, 1 (2%) was lostto follow-up, and 17 (38%) were alive, including 11/23 patients (50%)treated with 1×10⁶ cells/kg. Four patients received a second infusion ofanti-CD19 CAR T cells; one was in CR at 15 months post-re-dosing, 2 hadrelapsed by the month-3 assessment, and 1 withdrew consent prior to thefirst response assessment.

Clinical Pharmacology

CAR T-cell levels measured by CAR gene copies per μg DNA in blood peaked7-14 days post-anti-CD19 CART cells infusion for most patients andremained detectable in 2/12 evaluable patients at 12 months, both ofwhom were in CR (FIG. 7A; Table 22).

TABLE 22 CAR gene copies in blood over time CAR Gene 1 × 10⁶ 1 × 10⁶Copies per pg Original AE Revised AE DNA in Blood 2 × 10⁶ ManagementManagement 0.5 × 10⁶ Baseline (n = 6) (n = 14) (n = 9)  (n = 16) Median0 0 0 0 Range 0-0 0-0  0-0  0-0     Day 7 (n = 4) (n = 12) (n = 9)  (n =15) Median 62,411 154,386 91,287 3702 Range  11,097-162,97212,231-443,880     0-353,160 0-375,030 Week 2 (n = 5) (n = 14) (n = 8) (n = 13) Median 44,064 48,114 60,507 3669 Range   2228-106,110 7614-283,500 10,935-224,370  0-100,845 Week 4 (n = 5) (n = 11) (n = 9) (n = 13) Median 1304 3119 16,200 1588 Range  405-4860 1029-95,580  235-56,052 0-27,540  Week 8 (n = 0) (n = 5)  (n = 7) (n = 7) Median — 0527 219 Range — 0-907 0-972 0-9882   Month 3 (n = 4) (n = 11) (n = 6) (n= 9) Median 0 203 99 0 Range 0-0  0-1458 0-478 0-5508   Month 6 (n = 3)(n = 8)  (n = 0) (n = 7) Median 0 0 — 0 Range 0-0 0-105 — 0-518    Month9 (n = 1) (n = 6)  (n = 0) (n = 4) Median 0 0 — 0 Range 0-0 0-138 —0-0     Month 12 (n = 1) (n = 4)  (n = 0) (n = 3) Median 65 0 — 0 Range65-65 0-0  — 0-57    AE, adverse event; CAR, chimeric antigen receptor

CAR T cells were undetectable in the 5 patients with data available atrelapse. Median peak CAR T-cell levels were highest with 1×10⁶ CAR Tcells/kg and were similar between patients who received original vs.revised AE management (FIG. 7B; FIG. 8 ). Patients achieving CR/CRi hadgreater median peak expansion than non-responders, as did patients withundetectable vs. detectable MRD (FIG. 7C-D; FIG. 84B-C). Higher medianpeak expansion was also observed in patients with grade ≥3 vs. thosewith grade ≤2 NE (FIG. 7E-F; FIG. 8D-E). Of 13 patients who relapsed, 7had detectable CD19-positive cells at relapse, 3 had no detectableCD19-postive cells, and 3 had no data available.

Peak levels of key cytokines, chemokines, and pro-inflammatory markersoccurred by day 7, with some trending higher in patients dosed with2×10⁶ compared with 1×10⁶ CAR T cells/kg (IL-15, CRP, SAA, CXCL10,IFNγ), or lower in those with revised AE management vs those withoriginal AE management (IL-6, Ferritin, IL-1RA, IFNγ, IL-8, CXCL10,MCP-1) FIG. 9 ; FIG. 10 ). While peak IL-15 serum levels weresurprisingly lower in patients with grade ≥3 CRS, median peak levels ofseveral pro-inflammatory markers trended higher in patients with grade≥3 CRS and those with grade ≥3 NE (IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10,MCP-1, Granzyme B; FIG. 11 ).

Four patients tested positive during screening assays for anti-CARantibodies, but all were negative in confirmatory assays atleukapheresis. Characteristics of manufactured CAR T-cell products wereas anticipated and previously reported (Table 23).

TABLE 23 Product characteristics 1 × 10⁶ 1 × 10⁶ Median Original AERevised AE characteristic 2 × 10⁶ Management Management 0.5 × 10⁶(range) (n = 6) (n = 14) (n = 9) (n = 16) T-cell 32.9 (16.4-60.5) 41.1(9.9-73.2)  30.2 (0.1-65.0)  33.1 (12.5-80.9) subsets, % Naïve 34.5(15.1-42.7) 21.9 (14.6-40.7) 19.3 (3.2-36.3) 18.0 (3.0-48.2) Centralmemory Effector 8.9 (3.7-13.4) 8.9 (4.5-41.6) 14.5 (2.4-20.9) 14.3(2.4-38.1) Effector 20.7 (15.5-52.4) 18.4 (4.8-60.0)  19.9 (3.9-94.3)22.6 (1.0-45.3) memory CD4, % 44.7 (33.9-58.8) 47.6 (21.9-76.8)  56.8(41.0-93.7)  58.8 (28.5-85.9) CD8, % 55.4 (41.2-66.2) 49.0 (23.2-78.1)43.3 (6.3-59.0)  41.2 (14.1-71.4) CD4/CD8 0.8 (0.5-1.4)  1.0 (0.3-3.3)  1.4 (0.7-14.9) 1.4 (0.4-6.1) ratio IFNγ   7944.0 (1679.5-11214.4)  9980.5 (3025.0-37921.9)   10317.3 (5255.0-45235.7)   9059.5(1040.6-27859.1) production in co-culture (pg/mL)* *Co-cultureexperiments were performed using Toledo cells mixed in a 1:1 ratio withanti-CD19 CAR T cells product. IFNγ was measured in cell culture media24 h post-incubation using a qualified ELISA. AE, adverse event; IFNγ,interferon gamma

ZUMA-3 is the first multicenter study evaluating CAR T-cell therapy inadult R/R B-ALL to complete phase 1. In the phase 1 portion, noprotocol-defined DLTs were observed with anti-CD19 CAR T cells, and theAEs reported were consistent with prior studies of anti-CD19 CAR T-celltherapies. Neelapu S S. et al. N Engl J Med. 2017; 377(26):2531-2544;Maude S L et al. N Engl J Med. 2018; 378(5):439-448. The 1×10⁶ CAR Tcells/kg dose coupled with revised AE management guidelines had the mostfavorable risk/benefit ratio without compromising activity. Althoughpatients had high disease burden and were heavily pretreated, high ratesof remission and undetectable bone marrow MRD were achieved,particularly in those treated at the 1×10⁶ dose level; the ORR was 83%including 61% CR and 22% CRi, all of whom had undetectable MRD. Based onthese results showing that anti-CD19 CAR T cells are safe and havepromising efficacy, the 1×10⁶ CAR T cells/kg dose was chosen for furtherevaluation in phase 2 of ZUMA-3.

Use of anti-CD19 CAR T cells to treat adult R/R B-ALL has provendifficult owing to the highly proliferative nature of this disease andinability to tolerate treatment-related AEs. A previous CAR T-cell trialin this population was closed early due to fatal NE, including 5 casesof cerebral edema. DeAngelo D J, Ghobadi A, Park J H, et al. Journal forImmunoTherapy of Cancer. 2017; 5(Suppl 2):P217. Under the original AEmanagement guidelines in ZUMA-3, 2 patients died from grade 5 AEsconsidered related to anti-CD19 CAR T cells either secondary to CRS orin the context of CRS and NE outside the DLT-assessment timeframe. Inaddition to evaluating multiple doses to identify the dose with the mostmanageable toxicities, revised AE management guidelines requiringearlier steroid intervention for neurotoxicity and the use oftocilizumab only for CRS were implemented among 9 patients enrolled atthe 1×10⁶ CAR T cells/kg dose level. This resulted in a shorter durationof CRS events and lower incidence, severity, and duration of NE comparedwith 14 patients treated at the same dose under the original guidelines.

At a median follow-up of 22.1 months, responses were ongoing in 26% ofpatients, most of whom received 1×10⁶ CAR T cells/kg (32% ongoingCR/CRi). Responses tended to occur early after treatment. Most occurredwithin the first month, though 1 patient with extramedullary diseaseachieved CR at month 6. High response rates were observed across allprespecified subgroups, including a 100% CR rate in patients with Ph+disease. Response (CR/CRi) was associated with higher expansion of CARTcells measured within 2 weeks post-treatment. Similarly, in asingle-center, phase 1 study using an anti-CD19 CAR T-cell therapy alsocontaining a CD3ζ and CD28 co-stimulatory domain (Park J H et a. N EnglJ Med. 2018; 378(5):449-459), the overall CR rate was 83%, althoughpost-bridging therapy, only half of the patients had ≥5% blasts in thebone marrow, 28% had MRD, and 11% had undetectable MRD. Nevertheless,those trial results largely paralleled those of the present study,further supporting the potential utility of anti-CD19 CAR T-celltherapies using a CD3ζ and CD28 co-stimulatory domain in adult R/RB-ALL.

Tisagenlecleucel, an anti-CD19 CAR T-cell therapy containing a CD3ζT-cell activation domain and a 4-1BB co-stimulatory domain, is approvedfor the treatment of R/R B-ALL in children and young adults (≤25 years).Maude S L et al. N Engl J Med. 2018; 378(5):439-448; KYMRIAH(tisagenlecleucel) [package insert]. Novartis. East Hanover, N.J.; 2018.The dosing regimen for tisagenlecleucel in younger patients, however,resulted in substantial toxicity and CRS-related deaths in adults withR/R B-ALL. Frey Nev. et al. J Clin Oncol. 2020; 38(5):415-422. In asingle-center study in adult R/R B-ALL across two clinical trials,administering the dose in fractions resulted in manageable CRS and a 90%CR rate. Frey Nev. et al.. J Clin Oncol. 2020; 38(5):415-422. Similar toZUMA-3 observations, optimized dosing and toxicity management strategiesmay enable patients vulnerable to life-threatening treatment-relatedtoxicities to benefit from CAR T-cell therapy.

Despite differences in trial designs, patient populations, and OSmethodology, the median OS with 1×10⁶ CAR T cells/kg in the presentstudy was 16.1 months, whereas the median OS previously reported withblinatumomab, which also targets CD19, was 6.1-7.7 months in adult R/RB-ALL. Topp Miss. et al. Lancet Oncol. 2015; 16(1):57-66; Kantarjian H.et al. N Engl J Med. 2017; 376(9):836-847. Of 10 patients evaluable atrelapse for CD19 blast expression, 3 showed lack of CD19 expression,reminiscent of other reports attributing target-loss to selection ofexon splice variants and mutations. Sotillo E et al. Cancer Discov.2015; 5(12):1282-1295. In the present study, only 1/8 patients (13%)with blinatumomab as last prior therapy responded to blinatumomab. Thismay suggest immunologic incompetence in unmanipulated T cells in somepatients with R/R ALL, possibly limiting the utility of bispecificT-cell engager therapy. Of the 21 patients with prior blinatumomab inany line, 12 (57%) achieved CR/CRi following anti-CD19 CAR T celltherapy. As previously reported (Shah B D. et al.. J Clin Oncol. 2018;36(suppl):abstr 7006), responses to anti-CD19 CART cells were similarregardless of prior blinatumomab exposure in patients with continuedCD19 positivity. In addition, 6 patients achieving CR underwent SCT andwere censored at the time of SCT; 3 remained in remission.

Adults with R/R B-ALL achieved high rates of CR and undetectable bonemarrow MRD with a tolerable safety profile after treatment withanti-CD19 CAR T cells. The successful manufacture for all enrolledpatients and the relatively rapid turnaround time supported thefeasibility of providing this cellular-therapy treatment to patientswith rapidly progressing disease who need prompt treatment. By carefullyevaluating a range of doses and adopting safety strategies, includinguse of tocilizumab or steroids and conditions under which they should beadministered to manage AEs, it was possible to transition the study fromphase 1 to an international phase 2 study. There were no fatal cerebraledema cases in phase 1, a limitation of prior studies in thispopulation. Phase 2 of ZUMA-3 was ongoing at the 1×10⁶ CAR T cells/kgdose with revised AE management guidelines.

Example 10

This Example described the results of CD19ΔTyr260 in CD19 in B-ALLassociated with resistance to a CAR T cell therapy treatment. Afterfailing several therapies, including blinatumomab prior to KTE-X19, aB-ALL patient received a target dose of 1×10⁶ CAR T cell/kg. The patientdid not respond clinically; no CAR T and CD19-expressing lymphocyteswere detectable at Day 28. Peripheral blood mononuclear cells (PBMCs)were collected the patient with B-ALL pre- and post-KTE-X19 infusion atvarious time points. Multicolor flow cytometry was used to examine CD19(clone FMC63, HIB19, SJ25C1) surface expression on patient PBMCs andJurkat cell lines engineered to be CD19-wildtype (WT) or to expressCD19ΔTyr260. The presence of genetic variants was assessed usingenhanced whole genome and RNA sequencing (TruSeq Stranded Total RNA).Location of cellular protein expression was assessed using Western blotwith and without deglycosylating enzymes.

While local pathology concluded that preinfusion B lymphoblasts wereuniformly CD19^(dim), additional analyses of the same sample with FMC63(the single-chain variable fragment of KTE-X19) revealed that CD19 wasnot detectable in preinfusion B lymphoblasts. Results of RNA sequencingshowed an in-frame deletion within the intracellular domain of CD19 atTyr260 (CD19ΔTyr260) in circulating leukemia blasts. Additional analysisusing flow cytometry showed that CD19 expression was not detected onJurkat CD19ΔTyr260 cells but was present on Jurkat CD19-WT cells, whichsuggested that the lack of visualization of cells carrying this pointmutation and their resistance to CAR T cell therapy. Longitudinal RNAand DNA sequencing analysis showed that the mutation had occurred priorto infusion of CAR-T therapy. Fractionated cellular lysates showed WTCD19 in the cell membrane having a high and lower molecular weight band,as well as CD19ΔTyr260 expressed on the surface with a single lowmolecular weight band. Under deglycosylating conditions, only 1 band waspresent in both WT CD19 and CD19ΔTyr260 cellular fractions. Withoutbeing bound to any scientific theories or hypotheses, it is likely thatthe CD19ΔTyr260 mutation may result in lack of suitable or functionalCD19 glycosylation and/or inhibiting detection. The mutation in B-ALLmalignant cells may have potential implications for other anti-CD19 CARor non-CD19 CAR cell therapy.

Example 11

Patients with MCL who progress after BTKi therapy typically have a poorprognosis, with an overall survival of only 5.8 months with salvagetherapies. Martin P, et al. Blood. 2016; 127:1559-1563. In Phase 2ZUMA-2 study, KTE-X19 was evaluated in patients with MCL who were R/R to1-5 prior therapies, including a BTKi. Wang M, et al. N Engl J Med.2020; 382:1331-1342. At a median follow-up of 12.3 months, the ORR was93% (67% complete responses) in the primary efficacy analysis of ZUMA-2(N=60). Aggressive disease variants, including blastoid or pleomorphicMCL, are generally associated with poor clinical outcomes, yet ORR wascomparable across patients with various histologies in ZUMA-2. Wang M,et al. N Engl J Med. 2020; 382:1331-1342; Jain P and Wang M. Am JHematol. 2019; 94:710-725. In this study, the pharmacological profileand clinical outcomes in patient subgroups defined by MCL morphology andprior BTKi exposure in ZUMA-2 were compared, accompanied by acharacterization of product attributes and other pre-treatment factors.Patients underwent leukapheresis and conditioning chemotherapy followedby a single infusion of CD19 CAR-T cells at a target dose of 2×10⁶ CAR Tcells/kg, by single IV infusion on Day 0. Some patients receivedbridging therapy with dexamethasone (20-40 mg or equivalent PO or IVdaily for 1-4 days), ibrutinib (560 mg PO daily), or acalabrutinib (100mg PO twice daily), administered after leukapheresis and completed ≤5days before initiating conditioning chemotherapy; PET-CT was requiredpost-bridging. Primary endpoint was objective response rate (ORR[complete response (CR)+partial response]). Secondary endpoints wereduration of response (DOR), progression-free survival (PFS), OS,frequency of adverse events (AEs), levels of CAR T cells in blood, andlevels of cytokines in serum. Efficacy and safety analyses included allpatients who received CD19 CAR-T cell therapy. The first tumorassessment was done on Day 28. Bone marrow biopsy was done at screening,and if positive, not done, or indeterminate, a biopsy was needed toconfirm CR.

Of the 60 patients in ZUMA-2 with MCL treated with KTE-X19 with a medianfollow-up of 12.3 months, there was a 93% ORR, 67% CR rate, and 57% ofall patients and 78% of patients in CR had ongoing responses. CRS andneurologic events were mostly reversible (N=68 treated patients). About15% had Grade ≥3 CRS, 31% Grade ≥3 neurologic events, and 2 Grade 5 AEs(1 KTE-X19-related). Patient subgroups were defined by morphologicalcharacteristics (classical, blastoid, or pleomorphic MCL) and by priorexposure to ibrutinib only, acalabrutinib only, or both ibrutinib andacalabrutinib. Table 24. Baseline characteristics were generallycomparable across these groups. There was a trend toward higherpre-treatment tumor burden in patients previously treated withibrutinib. Product attributes, CAR T cell levels in blood, and cytokinelevels in serum were analyzed using previously described methods. LockeFla., et al. Mol Ther. 2017; 25:285-295. Product T cell attributes weregenerally comparable across MCL morphology subgroups. There were trendstoward increased product co-culture IFN-γ and percentage of CCR7+ cellsin products from patients with pleomorphic morphology. Table 25. ProductT cell Attributes were also generally comparable across prior BTKisubgroups. There was a trend toward increased product co-culture IFN-γin patients previously treated with ibrutinib. Table 26.

TABLE 24 Patient baseline characteristics. Ibrutinib Acalabrutinib Both(n = 52) (n = 10) (n = 6) Median age (range), years 65 (45-79) 57(38-73) 62 (55-72) ≥65 years, n (%) 32 (62) 4 (40) 3 (50) Male, n (%) 43(83) 9 (90) 5 (83) Stage IV disease, n (%) 44 (85) 9 (90) 5 (83) ECOG0/1, n (%) 52 (100) 10 (100) 6 (100) Median tumor burden^(a) 2697(386-16878) 1144 (293-14390) 536 (260-1174) (range), mm² Ki-67proliferation index, n/N (%) ≥50 25/38 (66) 3/5 (60) 6/6 (100) <50 13/38(34) 2/5 (40) 0 MCL morphology Classical 30 (58) 6 (60) 4 (67)Pleomorphic 1 (2) 2 (20) 1 (17) Blastoid 12 (23) 3 (30) 2 (33) Bonemarrow 28 (54) 3 (30) 6 (100) involvement, n (%) Extranodal disease, n(%) 31 (60) 3 (30) 4 (67) Median no. prior 3 (1-5) 3 (2-5) 3 (3-4)therapies (range) Prior bendamustine, n 28 (54) 7 (70) 2 (33) (%) ^(a)Asmeasured by the sum of product dimensions of all target lesions atbaseline. For subjects who had bridging therapy, the measurement afterbridging therapy is used as baseline.

TABLE 25 Cell characterizations and MCL morphology. MCL MorphologyClassical Blastoid Pleomorphic Median (range) (n = 40) (n = 17) (n = 4)Transduction rate, % 58.1 (35.0-82.4)  60.0 (46.0-79.4)  61.9(50.0-77.1) CD4/CD8 ratio 0.7 (0.04-2.8)^(a) 0.6 (0.2-1.1)^(a)  0.7(0.5-2.0)  CCR7+ T cells, % 40.0 (2.6-88.8)^(a)  35.3 (14.3-73.4)^(a)80.8 (57.3-88.8) CCR7− effector + effector 59.9 (11.1-97.4)^(a) 64.8(26.6-85.7)^(a) 19.2 (11.1-42.7) memory T cells, % (CCR7+ Tcells)/(CCR7− 0.7 (0.03-8.0)^(a) 0.5 (0.2-2.8)^(a)  4.7 (1.3-8.0) effector + effector memory T cells) ratio IFN-γ by coculture, pg/mL  6309.5 (424.0-2.0 × 10⁴)   6510.0 (2709.0-1.8 × 10⁴)   7687.5(424.0-1.2 × 10⁴) ^(a)Based on available data: classical, n = 38;blastoid, n = 16

TABLE 26 Cell characterizations and BTKi subgroups. BTKi ExposureIbrutinib Acalabrutinib Both Median (range) (n = 52) (n = 10) (n = 6)Transduction rate, % 56.7 (32.0-82.4)  65.0 (35.0-74.0) 58.5 (46.0-67.0)CD4/CD8 ratio 0.7 (0.04-3.7)^(a) 0.6 (0.3-1.2)  1.0 (0.7-1.9)  CCR7+ Tcells, % 39.3 (2.6-86.4)^(a)  42.7 (16.3-88.8) 49.5 (14.3-83.0) CCR7−effector + effector memory 60.6 (13.7-97.4)^(a) 57.3 (11.1-83.8) 50.6(17.0-85.7) T cells, % (CCR7+ T cells)/(CCR7− effector + 0.7(0.03-6.3)^(a) 0.8 (0.2-8.0)  1.2 (0.2-4.9)  effector memory T cells)ratio IFN-γ by coculture, pg/mL   6496.0 (424.0-2.0 × 10⁴)   5972.5(2502.0-1.8 × 10⁴)   7985.5 (2709.0-1.2 × 10⁴) ^(a)Based on availabledata: ibrutinib, n = 49

High rates of response were achieved across MCL morphology and priorBTKi subgroups. Table 27. Clinical benefit from KTE-X19 treatment wasobserved in all subgroups defined by MCL morphology or prior BTKi. Atrend toward a higher ongoing response rate at 6 months was observed inpatients previously treated with ibrutinib. Table 27. CRS andneurological events were generally comparable across MCL morphology andprior BTKi subgroups. Table 28. A trend toward increased rate of Grade≥3 neurological events was observed in patients with non-blastoidmorphology or previously treated with ibrutinib. Table 28.

TABLE 27 Rate of response MCL Morphology BTKi Exposure ClassicalBlastoid Pleomorphic Ibrutinib Acalabrutinib Both (n = 35) (n = 14) (n =4) (n = 45) (n = 9) (n = 6) ORR, n (%) 32 (91)¹ 13 (93)¹ 4 (100)¹ 43(96) 7 (78) 6 (100) CR, n (%) 22 (63)¹ 9 (64)¹ 3 (75)¹ 30 (67) 4 (44) 6(100) Ongoing response 18 (51) 8 (57) 3 (75) 25 (56) 3 (33) 6 (100) at 6mo, n (%) 12-mo OS, % 85.7 (69.0-93.8)¹ 71.4 (40.6-88.2)¹ 100.0 (NE-NE)¹82.0 (67.2-90.6) 77.8 (36.5-93.9) 100.0 (NE-NE) (95% CI) ¹Wang M, et al.N Engl J Med. 2020; 382: 1331-1342. CR, complete response; MCL, mantlecell lymphoma; NE, not evaluable; ORR, objective response rate; OS,overall survival

TABLE 28 Adverse Events MCL Morphology BTKi Exposure Classical BlastoidPleomorphic Ibrutinib Acalabrutinib Both n (%) (n = 40) (n = 17) (n = 4)(n = 52) (n = 10) (n = 6) CRS Any grade 36 (90) 15 (88) 4 (100) 50 (96)6 (60) 6 (100) Grade ≥3 6 (15) 1 (6) 1 (25) 9 (17) 1 (10) 0 Neurologicevents Any grade 25 (63) 11 (65) 3 (75) 33 (63) 4 (40) 6 (100) Grade ≥315 (38) 3 (18) 2 (50) 16 (31) 1 (10) 4 (67)

Comparisons across subgroups were conducted using the Kruskal-Wallistest; Dunn's post-hoc test was used to compare between groups.Pharmacological profile, product attributes, and safety data werereported for all 68 patients treated with KTE-X19 (2×10⁶ cells/kg). Thepharmacological and pharmacodynamic profile of KTE-X19 across MCLmorphology subgroups suggested increased CAR T cell expansion and selectpro-inflammatory cytokines in patients with classical morphologycompared with patients with blastoid morphology (FIGS. 12 and 13 ) or inpatients previously treated with ibrutinib compared with acalabrutinibalone FIGS. 14 and 15 ). Pre-treatment patient and productcharacteristics were generally comparable across MCL morphologies andsubsets with different prior therapies. Patients with blastoidmorphology exhibited decreased CAR T cell expansion, circulatingmyeloid-related cytokines and chemokines, and rate of Grade ≥3 CRS andneurologic events, while the clinical efficacy was comparable with thatof patients with classical morphology. The trend towards an improvedsafety profile in patients with blastoid morphology was commensuratewith lower peak CAR T cell expansion and decreased peak levels ofcytokines associated with myeloid-related inflammation. Patientspreviously treated with ibrutinib exhibited increased CAR T cellexpansion, circulating inflammatory cytokines and chemokines, and rateof Grade ≥3 neurologic events; as well as increased ongoing responserate at 6 months and an ORR comparable with that of patients previouslytreated with acalabrutinib alone. Patients previously treated withacalabrutinib exhibited decreased CAR T cell expansion and circulatingT1-related cytokines and chemokines, which was consistent with animproved safety profile.

Example 12

This example characterized two anti-CD19 CAR T therapies, KTE-X19prepared according to Example 5 and axicabtagene ciloleucel. Cells werelabeled with fluorescently-conjugated antibodies to CD3 (pan T cellmarker), CD14, CD19 (B cell marker), CD45 (pan-leukocyte marker), andCD56 (activation and NK marker) and assessed by flow cytometry. Cellviability was assessed using negative staining of a viability dye (SYTOXnear-IR). The lower limit of quantification (LLOQ) of the assay was 0.2%and for NK cells and monocytes was 5%. The percentage of NK cells wasdetermined (NK cells were CD45⁺, CD14⁻, CD3⁻, and CD56⁺; T cells wereCD45⁺, CD14⁻, and CD3⁻). The median percentages of NK cells from 23 lotsof axicabtagene ciloleucel and 97 lots of KTE-X19 were 1.9% (range0.8%-3.2%) and 0.1% (range 0.0%-2.8%), respectively. The medianpercentage of CD3⁻ cellular impurities from the same lots ofaxicabtagene ciloleucel and KTE-X19 were 2.4% (range 0.9%-4.6%) and 0.5%(range 0.3%-3.9%), respectively. The results of KTE-X19 and axicabtageneciloleucel in cell viability were ≥72% and ≥80%, respectively; inanti-CD19 CAR expression were ≥24% and ≥15%, respectively; in IFN-γproduction were ≥190 pg/mL and ≥520 pg/mL, respectively; and inpercentage of CD3⁺ cells were ≥90% and ≥85%, respectively.

Example 13

Additional results of patients receiving 2×10⁶ KTE-X19 cells/kg in asingle infusion in prior examples including EXAMPLE 2 and EXAMPLE 7 wereprovided. ORR by IRRC assessment was 92% (95% CI, 82-97) and CR Rate was67% (95% CI, 53-78). At a median follow-up of 17.5 months (range,12.3-37.6), 29 patients remained in ongoing responses. Ongoing responserates were largely consistent among patients with high-risk diseasecharacteristics. The first 28 patients treated had a median follow-up of32.3 months (range, 30.6-37.6). 39% of patients remained in continuedremission with no further therapy. In all enrolled patients (N=74), ORRwas 84% (59% CR rate). The medians for DOR, PFS, and OS were not reachedafter a median follow-up of 17.5 months. Table 29. The ongoing responserate was consistent across adverse prognostic groups. FIG. 16 . At amedian follow-up of 17.5 months, the ZUMA-2 study continued to showsubstantial and durable clinical benefit of KTE-X19 therapy in patientswith R/R MCL. No new safety signals were observed with additionalfollow-up. No new CRS or new Grade 5 events occurred since the previousreports. Table 30. AE rates decreased over time. KTE-X19 therapy showeda manageable safety profile with extended follow-up.

TABLE 29 Duration of response, progression-free survival, and overallsurvival. DOR PFS OS Median 15-Mo Rate Median 15-Mo Rate Median 15-MoRate (95% CI), mo (95% CI) (95% CI), mo (95% CI) (95% CI), mo (95% CI)Evaluable NR (13.6-NE)^(a) 58.6 (42.5-71.7)^(a) NR (9.6-NE) 59.2(44.6-71.2) NR (NE, NE) 76.0 (62.8, 85.1) pts (N = 60) Pts in CR NR(14.4-NE) 69.7 (49.3-83.2) NR (15.3-NE) 75.1 (56.8-86.5) NR (NE, NE)91.7 (76.2, 97.2) (n = 40) Pts in PR 2.2 (1.4-4.3) 24.1 (5.9-48.9) 3.1(2.3-5.2) 24.1 (5.9-48.9) 12.6 (3.3, NE) 46.7 (21.2, 68.7) (n = 15)^(a)Out of 55 total responding patients.

TABLE 30 Safety Analysis All Treated Patients (N = 68) On/After On/After3 Months 6 Months Post-Infusion Post-Infusion Any Grade Any Grade AE, n(%)^(a) Grade >3 Grade >3 Any AE 55 (81) 33 (48) 49 (72) 25 (37) Anemia22 (32)  9 (13) 13 (19) 4 (6) Neutropenia 20 (29) 16 (24) 14 (21) 11(16) Thrombocytopenia 20 (29) 14 (21) 14 (21)  9 (13) White blood cellcount decrease 16 (24)  9 (13) 12 (18) 6 (9) Fatigue 10 (15) 0 10 (15) 0Pneumonia  9 (13) 5 (7) 6 (9) 4 (6) Cough  8 (12) 0  7 (10) 0Hypogammaglobulinemia  8 (12) 0  7 (10) 0 Upper respiratory tractinfection  7 (10) 2 (3) 5 (7) 1 (1) ^(a)Includes AEs of any gradeoccurring in ≥10% of patients.

Of 57 efficacy-evaluable patients with data available, 48 (84%) haddetectable B cells at baseline. Among patients with ongoing responses at12 months, more than 50% of evaluable patients had detectable B cellsand gene-marked CAR T cells at months 6, 12, 15, and 24. Among patientsin ongoing response at 12 months, the percentage of patients withgene-marked CAR T cells generally decreased over time, with 100%, 93%,82%, 89%, 80%, and 56% at 3, 6, 12, 15, 18, and 24 months, respectively.There was a decreased CAR T cell peak expansion in patients that failedto respond to KTE-X19. Peak CAR T cell expansion was increased inpatients with an ongoing response at 12 months or in those who relapsedat 12 months, compared to nonresponding patients. Elevated CAR T celllevels were initially observed in patients who later relapsed, possiblypointing to alternate mechanisms for secondary treatment failure. CAR Tcell peak levels normalized by baseline tumor burden and ongoingresponse at 12-month data cut are shown in FIGS. 17A (INV) and 17B(CEN).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure.

We claim:
 1. A method for treating mantle cell lymphoma (MCL) or B cellALL in a subject in need thereof comprising administering to the subjecta therapeutically effective amount of a T cell product comprisingautologous T cells expressing an anti-CD19 chimeric antigen receptor(CAR).
 2. The method of claim 1, wherein the MCL and B cell ALL arerelapsed or refractory MCL and B cell ALL, optionally wherein the MCL isclassical, blastoid, and pleomorphic MCL.
 3. The method of any one ofclaims 1 and 2, wherein the MCL and B cell ALL is refractory to, or hasrelapsed following, one or more of chemotherapy, radiotherapy,immunotherapy (including a T cell therapy and/or treatment with anantibody or antibody-drug conjugate), an autologous stem celltransplant, or any combination thereof.
 4. The method of any one ofclaims 1 through 3, wherein the subject has received 1-5 priortreatments, optionally wherein at least one of the prior treatments isselected from autologous SCT, anti-CD20 antibody, anthracycline- orbendamustine-containing chemotherapy, and/or a Bruton Tyrosine Kinaseinhibitor (BTKi).
 5. The method of claim 4, wherein the BTKi isibrutinib or acalabrutinib.
 6. The method of any one of claims 1 through5, wherein R/R B cell ALL is defined as refractory to first-line therapy(i.e., primary refractory), relapsed ≤12 months after first remission,relapsed or refractory after ≥2 prior lines of systemic therapy, orrelapsed after allogeneic stem cell transplant (SCT), optionally,wherein the subject is required to have ≥5% bone marrow blasts, anEastern Cooperative Oncology Group performance status of 0 or 1, and/oradequate renal, hepatic, and cardiac function.
 7. The method of any oneof claims 1 through 6, wherein if the B cell ALL subject has receivedprior blinatumomab, the subject is required to have leukemic blasts withCD19 expression ≥90%.
 8. The method of any one of claims 1 through 7,wherein the subject receives bridging therapy after leukapheresis andbefore conditioning/lymphodepleting chemotherapy.
 9. The method of anyone of claims 1 through 8, wherein the MCL subject receives alymphodepleting chemotherapy regimen of cyclophosphamide 500 mg/m²intravenously and fludarabine 30 mg/m² intravenously, both given on eachof the fifth, fourth, and third days before T cell infusion.
 10. Themethod of any one of claims 1 through 9, wherein the B cell ALL subjectreceives a lymphodepleting regimen of fludarabine intravenous (IV) 25mg/m²/day on each of the fourth, third, second days before T cellinfusion, and cyclophosphamide IV 900 mg/m²/day on the second day beforeinfusion.
 11. The method of any one of claims 8 through 10, wherein theMCL bridging therapy is selected from dexamethasone (e.g., 20-40 mg orequivalent PO or IV daily for 1-4 days); methylprednisolone, ibrutinib(e.g., 560 mg PO daily), and/or acalabrutinib (e.g, 100 mg PO twicedaily); an immunomodulator; R-CHOP, bendamustine; alkylating agents;and/or platinum-based agents, wherein the bridging therapy isadministered after leukapheresis and completed in, for example, 5 daysor less before conditioning chemotherapy.
 12. The method of any one ofclaims 8 through 10, wherein the B cell ALL subject may receive any oneor more of the following bridging chemotherapy regimens: PredefinedBridging Chemotherapy Regimens Attenuated VAD Vincristine non-liposomal(1-2 mg IV weekly) or liposomal (2.25 mg/m² IV weekly), anddexamethasone 20-40 mg IV or PO daily × 3-4 days per week. Optionaldoxorubicin 50 mg/m² IV × 1 (first week only) Mercaptopurine 50-75mg/m²/day by mouth (administer at bedtime on an empty (6-MP) stomach toimprove absorption) Hydroxyurea Doses titrated between 15-50 mg/kg/day(rounded to the nearest 500 mg capsule and given as a single daily oraldose on a continuous basis) DOMP Dexamethasone 6 mg/m²/day PO (or IV)divided BID days 1-5, vincristine 1.5 mg/m² (maximum dose 2 mg) IV onday 1, methotrexate 20 mg/m² PO weekly, 6-MP 50-75 mg/m²/day PO dailyAttenuated Fludarabine 30 mg/m² IV days 1-2, cytarabine 2 g/m² IV days1-2, FLAG/FLAG-IDA G-CSF 5 μg/kg SC or IV starts on day 3 and cancontinue until day before the start of conditioning chemotherapy. Withor without idarubicin 6 mg/m² IV days 1-2 Mini-hyper CVAD Course A:Cyclophosphamide 150 mg/m² every 12 h × 3 days, (courses A and/ordexamethasone 20 mg/d IV or PO daily days 1-4 and 11-14, B) vincristine2 mg IV × 1 Course B: methotrexate 250 mg/m² IV over 24 hours on day 1,cytarabine 0.5 g/m² IV every 12 hours x 4 doses on days 2 and 3


13. The method of any one of claims 1 through 12, wherein the T cellproduct comprises CD4+ and CD8+ CAR T cells that are prepared fromperipheral blood mononuclear cells (PBMCs) by positive enrichment andconsequent partial or complete depletion of circulating cancer cells.14. The method of claim 13, wherein the PBMC are enriched for T cells bypositive selection for CD4+ and CD8+ cells, activated with anti-CD3 andanti-CD28 antibodies in the presence of IL-2, and then transduced with areplication-incompetent viral vector containing FMC63-28Z CAR, achimeric antigen receptor (CAR) comprising an anti-CD19 single-chainvariable fragment (scFv), CD28 and CD3-zeta domains.
 15. The method ofany one of claims 13 and 14, wherein the T cell product comprises fewercancer cells than a T cell product comprising T cells from aleukapheresis product that have not been positively selected for CD4+and CD8+ T cells.
 16. The method of any one of claims 13 through 15,wherein the T cell product has other superior product attributesrelative to a T cell product comprising T cells from a leukapheresisproduct that have not been positively selected/enriched for CD4+ andCD8+ T cells.
 17. The method of claim 16, wherein the superior productattributes are selected from increased percentage of CDRA45+CCR7+(naïve-like) T cells, decreased percentage of differentiated Tcells, increased percentage of CD3+ cells, decreased IFN-gammaproduction, decreased percentage of CD3− cells.
 18. The method of anyone of claims 1 through 17, wherein the MCL subject is administered oneor more doses of 1.8×10⁶, 1.9×10⁶, or 2×10⁶ CAR positive viable T cellsper kg body weight, with a maximum of 2×10⁸ CAR positive viable T cells(for patients 100 kg and above) and the B cell ALL subject isadministered 0.5×10⁶, 1×10⁶, or 2×10⁶ CAR positive viable T cells per kgbody weight, with a maximum of 2×10⁸ CAR positive viable T cells (forpatients 100 kg and above).
 19. The method of any one of claims 1through 17, wherein if the subject has achieved complete response to thefirst infusion, the subject may receive a second infusion of anti-CD19CAR T cells, if progressing following >3 months of remission, providedCD19 expression has been retained and neutralizing antibodies againstthe CAR are not suspected, wherein response is assessed using the Luganoclassification.
 20. The method of any one of claims 1 through 19,wherein the subject is monitored for signs and symptoms of cytokinerelease syndrome (CRS) and neurologic toxicity after T celladministration.
 21. The method of claim 20, wherein the subject ismonitored daily for at least seven days, preferably for four weeks,following infusion for signs and symptoms of CRS and neurologictoxicity.
 22. The method of any one of claims 20 and 21, wherein thesigns or symptoms associated with CRS include fever, chills, fatigue,tachycardia, nausea, hypoxia, and hypotension and the signs or symptomsassociated with neurologic events include encephalopathy, seizures,changes in level of consciousness, speech disorders, tremors, andconfusion.
 23. The method of any one of claims 20 through 22, whereincytokine release syndrome in MCL subjects is managed in accordance withthe following protocol: CRS Grade Tocilizumab Corticosteroids Grade 1 Ifnot improving after 24 Not applicable. Symptoms require hours,administer tocilizumab symptomatic treatment only 8 mg/kg intravenouslyover 1 (e.g., fever, nausea, fatigue, hour (not to exceed 800 mg).headache, myalgia, malaise). Grade 2 Administer tocilizumab 8 Manage perGrade 3 Symptoms require and mg/kg intravenously over 1 if noimprovement respond to moderate hour (not to exceed 800 mg). within 24hours after intervention. Repeat tocilizumab every 8 startingtocilizumab. Oxygen requirement less than hours as needed if not Ifimproving, taper 40% FiO2 or hypotension responsive to intravenousfluids corticosteroids. responsive to fluids or low or increasingsupplemental dose of one vasopressor or oxygen. Limit to a maximum ofGrade 2 organ toxicity. 3 doses in a 24-hour period; maximum total of 4doses if no clinical improvement in the signs and symptoms of CRS. Ifimproving, discontinue tocilizumab. Grade 3 Per Grade 2 AdministerSymptoms require and methylprednisolone 1 respond to aggressive mg/kgintravenously intervention. twice daily or Oxygen requirement greaterequivalent than or equal to 40% FiO2 or dexamethasone (e.g., hypotensionrequiring high- 10 mg intravenously dose or multiple vasopressors every6 hours) until or Grade 3 organ toxicity or Grade 1, then taper Grade 4transaminitis. corticosteroids. If improving, manage as Grade
 2. If notimproving, manage as Grade
 4. Grade 4 Per Grade 2 AdministerLife-threatening symptoms. methylprednisolo Requirements for ventilatorne 1000 mg support or continuous veno- intravenously per venoushemodialysis day for 3 days. (CVVHD), or Grade 4 organ If improving,taper toxicity (excluding corticosteroids, and transaminitis). manage asGrade
 3. If not improving, consider alternate immunosuppres sants.


24. The method of any one of claims 20 through 23, wherein neurologictoxicity in MCL subjects is managed in accordance with the followingprotocol: Grading Assessment Concurrent CRS No Concurrent CRS Grade 2Administer tocilizumab per claim Administer dexamethasone 10 15 formanagement of Grade 2 mg intravenously every 6 CRS. hours until theevent is Grade If not improving within 24 hours after 1 or less, thentaper starting tocilizumab, administer corticosteroids. dexamethasone 10mg intravenously every 6 hours until the event is Grade 1 or less, thentaper corticosteroids. If still not improving, manage as Grade 3.Consider non-sedating anti-seizure medicines (e.g., levetiracetam) forseizure prophylaxis. Grade 3 Administer tocilizumab per claim Administer15 for management of Grade 2 dexamethasone 10 CRS. mg intravenously Inaddition, administer dexamethasone 10 every 6 hours. mg intravenouslywith the first dose of Continue dexamethasone use tocilizumab and repeatdexamethasone until the event is Grade 1 or dose every 6 hours. Continueless, then taper corticosteroids. dexamethasone use until the event isIf not improving, manage as Grade 1 or less, then taper Grade 4.corticosteroids. If improving, discontinue tocilizumab and manage asGrade
 2. If still not improving, manage as Grade 4 (below). Considernon-sedating anti-seizure medicines (e.g., levetiracetam) for seizureprophylaxis. Grade 4 Administer tocilizumab per claim Administer 15 formanagement of Grade 2 methylprednisolone 1000 CRS. mg intravenously perday Administer methylprednisolone 1000 mg for 3 days. intravenously perday with first dose of If improving, then manage as tocilizumab andcontinue Grade
 3. methylprednisolone 1000 mg If not improving,intravenously per day for 2 more days. consider alternate If improving,then manage as Grade
 3. immunosuppressants. If not improving, consideralternate immunosuppressants. Consider non-sedating anti-seizuremedicines (e.g., levetiracetam) for seizure prophylaxis.


25. The method of any one of claims 1 through 24, wherein the MCLsubject is a high-risk patient as determined by a Ki-67 tumorproliferation index ≥50% and/or presence of a TP53 mutation.
 26. Themethod of any one of claims 20 through 22, wherein CRS in a B cell ALLsubject is managed according to the following protocol: CRS GradeTocilizumab Corticosteroids Grade 1 If not improving after 24 Notapplicable. Symptoms require hours, administer tocilizumab symptomatictreatment only 8 mg/kg intravenously over 1 (e.g., fever, nausea,fatigue, hour (not to exceed 800 mg). headache, myalgia, malaise). Grade2 Administer tocilizumab 8 Manage per Grade 3 Symptoms require and mg/kgintravenously over 1 if no improvement respond to moderate hour (not toexceed 800 mg). within 24 hours after intervention. Repeat tocilizumabevery 8 starting tocilizumab. Oxygen requirement less than hours asneeded if not If improving, taper 40% FiO2 or hypotension responsive tointravenous fluids corticosteroids. responsive to fluids or low orincreasing supplemental dose of one vasopressor or oxygen. Limit to amaximum of Grade 2 organ toxicity. 3 doses in a 24-hour period; maximumtotal of 4 doses if no clinical improvement in the signs and symptoms ofCRS. If improving, discontinue tocilizumab. Grade 3 Per Grade 2Administer Symptoms require and methylprednisolone 1 respond toaggressive mg/kg intravenously intervention. twice daily or Oxygenrequirement greater equivalent than or equal to 40% FiO2 ordexamethasone (e.g., hypotension requiring high- 10 mg intravenouslydose or multiple vasopressors every 6 hours) until or Grade 3 organtoxicity or Grade 1, then taper Grade 4 transaminitis. corticosteroids.If improving, manage as Grade
 2. If not improving, manage as Grade 4.Grade 4 Per Grade 2 Administer Life-threatening symptoms.methylprednisolo Requirements for ventilator ne 1000 mg support orcontinuous veno- intravenously per venous hemodialysis day for 3 days.(CVVHD), or Grade 4 organ If improving, taper toxicity (excludingcorticosteroids, and transaminitis). manage as Grade
 3. If notimproving, consider alternate immunosuppres sants.


27. The method of any one of claims 20 through 22 and 26, whereinneurologic toxicity in a B cell ALL subject is managed in accordancewith one of the following two protocols: NE Revised Management GradeOriginal Management Guidelines Guidelines Grade 1 Supportive careSupportive care Neurological examination and Closely monitor neurologicadditional work-up as clinically status indicated Consider prophylacticantiepileptic Grade 2 Supportive Care and Evaluation Supportive Care andEvaluation Neurological examination, brain MRI, Continuous cardiactelemetry and evaluation of CSF; consider EEG as and pulse oximetry asclinically indicated indicated Consider prophylactic antiepilepticSerial neurological examinations to include fundoscopy and Glasgow ComaScore, brain MRI, evaluation of CSF, EEG; consider neurology consultAdminister antiepileptics for patients with seizures TocilizumabTocilizumab Consider tocilizumab 8 mg/kg IV over 1 For patients withhour (not to exceed 800 mg) for patients concurrent CRS, administer withcomorbid conditions (eg, grade >2 tocilizumab 8 mg/kg IV over CRS) 1hour (not to exceed 800 mg); repeat every 4-6 hours as needed if notresponsive to IV fluids or increasing supplemental oxygen, for a maximumof 3 doses in 24 hours Discontinue tocilizumab if patient improvesCorticosteroids Corticosteroids N/A For patients without concurrent CRS,administer dexamethasone 10 mg IV every 6 hours For patients withconcurrent CRS, if no improvement within 24 hours after startingtocilizumab, administer dexamethasone 10 mg IV every 6 hours Tapercorticosteroids if patient improves Grade 3 Supportive Care andEvaluation Supportive Care and Evaluation Per grade 2 Manage inmonitored care or Monitor with continuous cardiac ICU telemetry andpulse oximetry Tocilizumab Tocilizumab Consider tocilizumab 8 mg/kg IVover 1 Per grade 2 hour (not to exceed 800 mg); repeat Discontinuetocilizumab if every 4-6 hours if symptoms have not patient improvesstabilized or improved Corticosteroids Corticosteroids Considercorticosteroids (eg, Administer dexamethasone 10 dexamethasone 10 mg IVevery 6 hours mg IV every 6 hours or methylprednisolone l mg/kg BID)Taper corticosteroids if for worsening symptoms despite patient improvestocilizumab Grade Supportive Care and Evaluation Supportive Care andEvaluation Per grade 2 Per grade 3 Monitor with continuous cardiacMechanical ventilation may telemetry and pulse oximetry be requiredAdminister immunosuppresants if patient does not improve TocilizumabTocilizumab Administer tocilizumab per grade 3 if Per grade 2 notpreviously administered Corticosteroids Corticosteroids Administercorticosteroids (eg, Administer high-dose methylprednisolone ig/d × 3days, corticosteroids (eg, followed by 250 mg BID × 2 days, thenmethylprednisone ig/d × 3 125 mg BID × 2 days, then 60 mg BID × days) 2days) Taper corticosteroids if patient improves


28. The method of any one of claims 1 through 27, wherein the B cell ALLsubject may receive any one or more of the following bridgingchemotherapy regimens: Predefined Bridging Chemotherapy RegimensAttenuated VAD Vincristine non-liposomal (1-2 mg IV weekly) or liposomal(2.25 mg/m² IV weekly), and dexamethasone 20-40 mg IV or PO daily x 3-4days per week. Optional doxorubicin 50 mg/m² IV × 1 (first week only)Mercaptopurine (6- 50-75 mg/m²/day by mouth (administer at bedtime on anempty MP) stomach to improve absorption) Hydroxyurea Doses titratedbetween 15-50 mg/kg/day (rounded to the nearest 500 mg capsule and givenas a single daily oral dose on a continuous basis) DOMP Dexamethasone 6mg/m²/day PO (or IV) divided BID days 1-5, vincristine 1.5 mg/m²(maximum dose 2 mg) IV on day 1, methotrexate 20 mg/m² PO weekly, 6-MP50-75 mg/m²/day PO daily Attenuated Fludarabine 30 mg/m² IV days 1-2,cytarabine 2 g/m² IV days 1-2, FLAG/FLAG-IDA G-CSF 5 μg/kg SC or IVstarts on day 3 and can continue until day before the start ofconditioning chemotherapy. With or without idarubicin 6 mg/m² IV days1-2 Mini-hyper CVAD Course A: Cyclophosphamide 150 mg/m² every 12 h × 3days, (courses A and/or dexamethasone 20 mg/d IV or PO daily days 1-4and 11-14, B) vincristine 2 mg IV × 1 Course B: methotrexate 250 mg/m²IV over 24 hours on day 1, cytarabine 0.5 g/m² IV every 12 hours × 4doses on days 2 and 3


29. Autologous T cells expressing an anti-CD19 CAR for use in a methodfor treating mantle cell lymphoma (MCL) or B cell ALL according to anyone of claims 1 through
 28. 30. Use of autologous T cells expressing ananti-CD19 CAR in the manufacturing of a medicament for treating mantlecell lymphoma (MCL) or B cell ALL according to any one of claims 1through
 28. 31. A method of predicting: (i) objective response of asubject to a CAR T cell treatment (optionally, according to the methodof any one of claims 1 through 28) comprising measuring peak CAR T celllevels and comparing them to a reference standard, wherein objectiveresponse is positively associated with peak CAR T cell levels, whereinobjective response includes both complete response and partial response,and wherein all responses are assessed using the Lugano classification.(xii) minimal residual disease (e.g., at week 4) in response to a CAR Tcell treatment (optionally, according to the method of any one of claims1 through 28) comprising measuring peak CAR T cell levels and comparingthem to a reference standard, wherein negative minimal residual diseaseis associated with higher peak CAR T cell levels. (xiii) grade ≥3 CRSand/or grade ≥3 neurologic events (NE) in a subject receiving CAR T celltreatment (optionally, according to a method of any one of claims 1through 28) comprising measuring peak CAR T cell expansion aftertreatment and comparing the levels to a reference value, wherein thehigher the CAR T cell expansion, the higher the chance for grade ≥3 CRSand/or grade ≥3 NE events. (xiv) grade ≥3 CRS and/or grade ≥3 NEcomprising measuring the peak levels of GM-CSF and IL-6 post-CAR T celltreatment (optionally, according to the method of any one of claims 1through 28) and comparing them to a reference level, wherein the higherthe peak level of these cytokines, the higher the chance for grade ≥3CRS and/or grade ≥3 NE. (xv) grade ≥3 CRS in a subject receiving CAR Tcell treatment (optionally, according to a method of any one of claims 1through 28) comprising measuring the peak level of serum ferritinpost-CAR T cell treatment and comparing it to a reference level, whereinthe higher the peak level of ferritin, the higher the chance for grade≥3 CRS. (xvi) grade ≥3 CRS comprising measuring the peak levels of serumIL-2 and IFN-gamma post-CAR T cell treatment (optionally, of any one ofclaims 1 through 28) and comparing them to a reference level, whereinthe higher the peak level of IL-2 and IFN-gamma, the higher the chancefor grade ≥3 NE. (xvii) grade ≥3 CRS comprising measuring thecerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8,and/or vascular cell adhesion molecule (VCAM) post-CAR T cell treatment(optionally, of any one of claims 1 through 28) and comparing them to areference level, wherein the higher the cerebrospinal fluid levels ofC-reactive protein, ferritin, IL-6, IL-8, and/or vascular cell adhesionmolecule (VCAM), the higher the chance for grade ≥3 NE (xviii) grade ≥3CRS post-CAR T cell treatment (optionally, according to a method of anyone of claims 1 through 28) comprising measuring peak serum levels ofIL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a,MIP-1b, granzyme A, granzyme B, and/or perforin after anti-CD19 CAR Ttreatment and comparing the levels to reference levels, wherein the peakserum levels of IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10,IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and/or perforin associatepositively with grade ≥3 CRS. (xix) grade ≥3 CRS post-CAR T celltreatment of B cell ALL (optionally, according to a method of any one ofclaims 1 through 28) comprising measuring peak serum level of IL-15after anti-CD19 CAR T treatment and comparing the levels to referencelevels, wherein the peak serum level of IL-15 associates negatively withgrade ≥3 CRS. (xx) grade ≥3 CRS and/or grade ≥3 NE post-CAR T celltreatment (optionally, according to a method of any one of claims 1through 28) comprising measuring peak serum levels of IL-6, TNFα,GM-CSF, IL-10, MIP-1b, and granzyme B after anti-CD19 CAR T treatmentand comparing the levels to reference levels, wherein peak serum levelsof IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B associatepositively with grade ≥3 CRS and grade ≥3 NE. (xxi) whether a patient isgoing to be MRD (10⁻⁵ sensitivity) negative at 4 weeks/one monthpost-CAR T cell treatment (optionally, of any one of claims 1 through28), comprising measuring peak serum levels of IFN-γ, IL-6, and/or IL-2after treatment and comparing the level to a reference standard, whereinpeak serum levels of IFN-γ, IL-6, and/or IL-2 associate positively withbeing MRD negative at one month.
 32. The method of any one of claims 20through 24, 26, 27, and 30 through 31, wherein CRS and NE are graded bythe method described in Lee et al., Blood 2014; 124: 188-195.
 33. Themethod of claim 31, wherein the reference standard is established by anymethod generally used in the biomarker arts, such as quartile analysisof patient populations with known responses, grades of toxicity, and MRDlevels.
 34. The method of claim 31, wherein CAR T cell levels aremeasured by CAR gene copies per microgram of DNA in blood.
 35. Themethod of any one of claims 1 through 34, further comprising reducingthe levels/activity of the cytokines that associate positively withgrade ≥3 CRS and/or grade ≥3 NE post CAR T cell infusion to reduce grade≥3 CRS and/or grade ≥3 NE.
 36. A method of improving the effectivenessof CAR T cell treatment (e.g., of classical, blastoid, and pleomorphicMCL, and B cell ALL), in a subject in need thereof, comprisingmanipulating the T cell phenotype of the T cell product administered tothe subject, optionally wherein the manipulation comprises increasingthe number of CD3+ T cells, decreasing the number of CD3− cells,increasing the number/percentage of CDRA45+ CCR7+(naïve-like) T cellsand/or decreasing the number/percentage of differentiated cells in the Tcell product during production, decreasing the levels of IFN-gammaproduction by the T cells, wherein the improvement is observed relativeto the effectiveness of a T cell product that is prepared without anyintentional manipulation of the number/percentage of CDRA45+ CCR7+(naïve-like) T cells and/or the number/percentage of differentiatedcells in the T cell product.